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EP4355775A1 - Anticorps anti-ccl2 bispécifiques - Google Patents

Anticorps anti-ccl2 bispécifiques

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Publication number
EP4355775A1
EP4355775A1 EP22734270.6A EP22734270A EP4355775A1 EP 4355775 A1 EP4355775 A1 EP 4355775A1 EP 22734270 A EP22734270 A EP 22734270A EP 4355775 A1 EP4355775 A1 EP 4355775A1
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
acid sequence
domain comprises
ccl2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22734270.6A
Other languages
German (de)
English (en)
Inventor
Guy Georges
Jens Fischer
Lukasz KACPRZYK
Valeria RUNZA
Jasmin Sydow-Andersen
Cristina BERTINETTI-LAPATKI
Michael GERTZ
Shu Feng
Siok Wan GAN
Wei Shiong Adrian HO
Runyi Adeline LAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
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Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of EP4355775A1 publication Critical patent/EP4355775A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to bispecific anti-CCL2 antibodies binding to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancers, inflammatory, autoimmune and ophthalmologic diseases.
  • the CCL2/CCR2 axis is the main mediator of immature myeloid cell recruitment into the tumor.
  • CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM) building up a chemoattractant gradient.
  • ECM extracellular matrix
  • MDSCs myeloid-derived suppressive cells
  • MDSCs may reduce or even impair the efficacy of any T cell-activating therapy (Meyer et al, 2014).
  • CCL2 has also been implicated in the promotion of angiogenesis, metastasis and tumor growth, suggesting that neutralizing CCL2 might contribute to several lines of anti-tumor intervention.
  • CCL2 Targeting CCL2 -as opposed to its receptor- will specifically inhibit the undesired CCL2-mediated effects, sparing those that might signal through the same receptor (CCR2) but different ligands (e.g. CCL7, CCL8, CCL13) which are involved in the recruitment of other immune cell populations, like Thl and NK cells.
  • CCR2 CCR2
  • ligands e.g. CCL7, CCL8, CCL13
  • CCL2 has been a preferred antibody-target in several studies aiming at neutralizing its elevated levels caused by different inflammatory diseases, such as rheumatoid arthritis (Haringman et al, Arthritis Rheum. 2006 Aug;54(8):2387-92), idiopathic pulmonary fibrosis (Raghu et al, Eur Respir J. 2015 Dec;46(6): 1740-50), diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32: 307-315) and cancer (Sandhu et al, Cancer Chem other Pharmacol. 2013 Apr;71(4): 1041-50).
  • rheumatoid arthritis Hardingman et al, Arthritis Rheum. 2006 Aug;54(8):2387-92
  • idiopathic pulmonary fibrosis Rospir J. 2015 Dec;46(6): 1740-50
  • diabetic nephropathy Mienne et al, Nephrol Dial Transplant (2017) 32:
  • the present invention relates to certain bispecific anti-CCL2 antibodies binding to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancers, inflammatory, autoimmune and ophthalmologic diseases.
  • the present invention provides a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2 and a second different antigen-binding site that (specifically) binds to a second different epitope on human CCL2, wherein the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids), Hinge is a heavy chain hinge region,
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • the VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93; I) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:94.
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgG isotype, preferably of human IgGl isotype.
  • the bispecific antibody is not cross-reactive to other CCL homologs, (shows 100 time less binding to other CCL homologs (selected from the group of CCL8, CCL7, and CCL13) compared to the binding to CCL2
  • the bispecific antibody binds to the first and second epitope on human CCL2 in ion-dependent manner.
  • the bispecific antibody binds to human CCL2 in pH dependent manner and wherein the first antigen binding site and the second antigen binding site both bind to CCL2 with a higher affinity at neutral pH than at acidic pH.
  • the bispecific antibody binds to human CCL2 with a 10 times higher affinity at pH 7.4, than at pH 5.8.
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and/or iii) N434A (suitable for increasing affinity to FcRn for longer plasma half- life); and/or iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).
  • Kabat EU numbering i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and / P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life); and/ iv) Q438R and S440E(suitable for suppressing rheumatoid factor binding).
  • Rabat EU numbering i) Q311R and / P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T
  • the invention further provides an isolated nucleic acid encoding the bispecific antibody according to the invention as described herein.
  • the invention further provides a host cell comprising such nucleic acid .
  • the invention further provides a method of producing the bispecific antibody comprising culturing such host cell so that the bispecific antibody is produced.
  • the invention further provides a pharmaceutical formulation comprising the bispecific antibody according to the invention as described herein and a pharmaceutically acceptable carrier.
  • the invention further provides the bispecific antibody according to the invention as described herein for use as a medicament.
  • the invention further provides the bispecific antibody according to the invention as described herein for use in treating cancer.
  • the invention further provides the bispecific antibody according to the invention as described herein for use in treating an inflammatory or autoimmune disease.
  • the invention further provides the use of the bispecific antibody according to the invention as described herein in the manufacture of a medicament.
  • the invention is based, in part, on the finding that the bispecific antibodies as described herein use different anti-CCL2 antigen binding sites as first and second antigen binding site/moiety.
  • These bispecific anti-CCL2 antibodies bind to certain epitopes of CCL2 with high specificity, and have ability to specifically inhibit binding of CCL2 to its receptor CCR2. They show improved immune complex formation compared to monospecific antibodies and improved CCL2 abrogation in vivo.
  • the specific bispecific anti-CCL2 antibodies in the contorsbody format described herein show in addition valuable properties like low viscosity (which allows e.g. high concentration solutions suitable e.g. for subcutaneous administration)
  • 1A5 1G9
  • Figure 2a Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody CNT0888-SG1 (wild type IgGl) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody CNTO888-SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody CNT0888-SG1 (wild type IgGl)
  • Figure 2b Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 11K2- SG1 (wild type IgGl) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 2c Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody ABN912-SG1 (wild type IgGl) orb) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody ABN912- SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 2d Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A4- SG1 (wild type IgGl) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A4-SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 2e Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A5- SG1 (wild type IgGl) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A5-SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 2f Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1G9 - SGI (wild type IgGl) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1G9 -SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 2g Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 2F6- SG1 (wild type IgGl) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and monospecific anti-CCL2 antibody 20mg/kg 2F6-SG105 (Fc receptor binding silenced IgGl) into FcRn transgenic mice.
  • Figure 3 Shows the time course of serum total mouse CCL2 concentration ( Figure°3a) and antibody-time profile (Figure°3b) after i.v.
  • Figure 4a Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//1G9-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//1G9-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//1G9-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4b Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//11K2-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//11K2-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//11K2-WT IgGl (wild type IgGl with intact Fc receptor binding)
  • b) dotted line 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg bi
  • Figure 4c Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//1G9-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//1G9-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • a) solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//1G9-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20m
  • Figure 4d Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//1A5-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody CNT0888//1A5-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4e Serum concentration of hCCL2 over time after i.v.
  • pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 1 A5//1G9- WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti- CCL2 antibody 1A5//1G9-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4f Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//2F6- WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti- CCL2 antibody 11K2//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • a) solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 11K2//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4g Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody ABN912//11K2-WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: 0. lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody ABN912//11K2-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • a) solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody ABN912//11K2-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4h Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 1 A4//2F6- WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti- CCL2 antibody 1A4//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • a) solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 1 A4//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • Figure 4i Serum concentration of hCCL2 over time after i.v. injection of pre formed immune complex consisting of a) solid line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 1 A5//2F6- WT IgGl (wild type IgGl with intact Fc receptor binding) or b) dotted line: O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti- CCL2 antibody 1A5//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • a) solid line O.lmg/kg human CCL2 (hCCL2) and 20mg/kg bispecific anti-CCL2 antibody 1 A5//2F6-PGLALA (Fc receptor binding silenced IgGl) into Balb/c mice.
  • FIG. 5a Biacore® sensorgrams showing binding profile to monomeric CCL2 at pH7.4 (black line) and pH5.8 (grey line) of the four modified 11K2 and four CNT0888 variants, and the 16 bispecific anti-CCL2 antibodies CKLOOl to CKL016 resulting of the respective combination antigen binding moieties of the four modified 11K2 and four CNT0888 variants.
  • Figure 5b Biacore® sensorgrams showing binding profile to monomeric CCL2, of the four modified 11K2 and four CNT0888 variants, and the 16 bispecific anti-CCL2 antibodies CKLOOl to CKL016 resulting of the respective combination antigen binding moieties of the four modified 11K2 and four CNT0888 variants.
  • An additional dissociation phase at pH5.8 was integrated into the BIACORE® assay immediately after the dissociation phase at pH 7.4.
  • FIG. 6 Biacore® sensorgrams showing binding profile of bispecific anti-CCL2 antibodies CKLOOl, CKLO02, CKLO03 and CKLO04 to monomeric CCL8 at pH7.4 (black line) and pH5.8 (grey line).
  • Figure 7a Serum concentration of hCCL2 over time after injection of pre-formed immune complex consisting of hCCL2 and bispecific anti-CCL2 antibodies (parental CNTO//11K2 and pH dependent variants CKLOOl, CKLO02, CKLO03 and CKLO04) into SCID mice.
  • Figure 7b Serum concentration of hCCL2 over time after injection of pre-formed immune complex consisting of hCCL2 and CKLO03 (with IgGl wild type Fc) or CKL003-SG1099, (CKLO03 with enhanced pi Fc) into SCID mice.
  • CCL2-0048 the parent unmodified bispecific antibody CNT0888/l lk2k2 IgGl of CKL02, which is non-pH dependent, also shows an ICso of 0,2 pg/ml, since pH-dependency is critical for antigen sweeping, a phenomenon that does not take place in this assay.
  • the corresponding monospecific antibodies CNT0888 IgGl and humanized llk2 IgGl display ICso values of 0.3 and 0.7 pg/ml, respectively, while the huIgGl isotype control shows no inhibition (Fig 8, right panel).
  • Figure 9 In vivo anti-tumor activity in a genetically-modified mouse model.
  • Figure 10 Serum total (left) and free (right) CCL2 levels during the in vivo anti tumor activity study (see efficacy in Figure 9) under treatment with bispecific anti-CCL2 antibodies (vehicle in black, CKL02 wild type IgGl in grey, and pi-enhanced Fc (CKLO2-SG1099) in white bars/dotted line).
  • Figure 11 Proof of concept study of CCL2 sweeping efficiency in cynomolgus monkeys.
  • right panel individual concentration-time profile
  • Figure 12 Proof of concept study of CCL2 sweeping efficiency in cynomolgus monkeys.
  • Figure 13 Free CCL2 concentration-time profiles in serum of cynomolgus monkeys; left panel: average free CCL2 concentration-time profiles of the four antibodies is presented over seven days; right panel: individual free CCL2 concentration-time profile of individual 4 (group 2) is presented over the duration of the PK study (70 days); average profiles were calculated using a value of 0.01 ng/mL (lower limit of quantification) for samples that were below detection limit.
  • Figure 14 PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.
  • Figure 15 PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.
  • Figure 16 PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.
  • FIG 17 Exemplary scheme of a bispecific anti-CCL2 antibody of the present invention (in the socalled contorsbody (CB) format.
  • CB contorsbody
  • FIG. 18 SEC complex: SEC of CCL2 complex with the bi-paratopic antibodies P1AF8139 (abbreviated as 39) and P1AF8143 (abbreviated as 43)
  • FIG. 19A Y- shape bi-paratopic antibodies with the reference antibody PI AD8325
  • Fig. 19B contorsbodies P1AF8142 and PlAF8143 together with reference antibody P1AD8325
  • Fig. 20A Y- shape bi-paratopic antibodies with the reference antibody PI AD8325
  • Fig. 20B contorsbodies P1AF8142 and P1AF8143 together with reference antibody P1AD8325
  • the present invention relates to bispecific anti-CCL2 antibodies binding to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancers, inflammatory, autoimmune and ophthalmologic diseases.
  • bispecific anti-CCL2 antibodies comprises a first antigen-binding site that (specifically) binds to a first epitope on human CC2 and a second different antigen-binding site that (specifically) binds a different second epitope, wherein bispecific anti-CCL2 antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain.
  • CCL2 human CCL2
  • MCP-1 monocyte chemotactic protein 1
  • SMC-CF smooth muscle cell chemotactic factor
  • LDCF lymphocyte-derived chemotactic factor
  • GDCF glioma-derived monocyte chemotactic factor
  • TDCF tumor-derived chemotactic factors
  • HC11 human cytokine 11
  • MCAF monocyte chemotactic and activating factor
  • the gene symbol is SCYA2, the JE gene on human chromosome 17, and the new designation is CCL2 (Zlotnik, Yoshie 2000. Immunity 12: 121-127).
  • JE is the mouse homolog of human MCP- 1/CCL2.
  • wild type CCL-2 (wt CCL2) can exist as monomer but actually can also form dimers at physiological concentrations. This monomer-dimer equilibrium is certainly different and has to be carefully taken into account for all in vitro experiments described where different concentrations might be used.
  • we generated point mutated CCL2 variants The “P8A” variant of CCL2 carries a mutation in the dimerization interface resulting in an inability to form a dimer leading to a defined, pure CCL2 monomer.
  • the “T10C”“variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20; 135(11):4325-32).
  • the CCL2/CCR2 axis is the main mediator of immature myeloid cell recruitment into the tumor.
  • CCL2 is overexpressed by malignant cells and binds to the extracellular matrix (ECM) building up a chemoattractant gradient.
  • ECM extracellular matrix
  • MDSCs myeloid-derived suppressive cells
  • MDSCs may reduce or even impair the efficacy of any T cell-activating therapy (Meyer et al, 2014).
  • CCL2 has also been implicated in the promotion of angiogenesis, metastasis and tumor growth, suggesting that neutralizing CCL2 might contribute to several lines of anti-tumor intervention.
  • CCL2 - as opposed to its receptor - will specifically inhibit the undesired CCL2-mediated effects, sparing those that might signal through the same receptor (CCR2) but different ligands (e.g. CCL7, CCL8, CCL13) which are involved in the recruitment of other immune cell populations, like Thl and NK cells.
  • CCR2 CCR2
  • ligands e.g. CCL7, CCL8, CCL13
  • CCL2 has been a preferred antibody-target in several studies aiming at neutralizing its elevated levels caused by different inflammatory diseases, such as rheumatoid arthritis (Haringman et al, 2006), idiopathic pulmonary fibrosis (Raghu et al, 2015), diabetic nephropathy (Menne et al, 2016) and cancer (Sandhu et al, 2013).
  • rheumatoid arthritis Hardingman et al, 2006
  • idiopathic pulmonary fibrosis Rosu et al, 2015
  • diabetic nephropathy Mienne nephropathy
  • cancer Sandhu et al, 2013
  • KD antibody-antigen dissociation constants
  • CCL2 neutralization appears to be more obviously relevant in patients with elevated serum levels of CCL2, which has been observed in several types of cancers like breast cancer (BC), ovarian cancer (OvCa), colorectal cancer (CRC), pancreatic cancer and prostate cancer.
  • BC breast cancer
  • OvCa ovarian cancer
  • CRC colorectal cancer
  • pancreatic cancer pancreatic cancer
  • PC breast cancer
  • OvCa ovarian cancer
  • CRC colorectal cancer
  • prostate cancer pancreatic cancer
  • patients within these indications who do not present this serology but whose tumors are highly infiltrated with immune cells of the myeloid lineage might very well profit from this novel therapy due to the many roles that CCL2 plays in the tumor context as mentioned above.
  • an antibody “binding to human CCL2”, “specifically binding to human CCL2”, “that binds to human CCL2” or “anti-CCL2 antibody” refers to an antibody specifically binding to the human CCL2 antigen with a binding affinity of a KD-value of 5.0 x 10 8 mol/1 or lower, in one embodiment of a KD-value of 1.0 x 10 9 mol/1 or lower, in one embodiment of a KD-value of 5.0 x 10 8 mol/1 to 1.0 x 10 13 mol/1.
  • binding affinity is determined with a standard binding assay, such as surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala, Sweden) e.g. using constructs comprising CCL2 extracellular domain (e.g. in its natural occurring 3 dimensional structure).
  • binding affinity is determined with a standard binding assay using exemplary soluble CCL2.
  • Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • monospecific antibody denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.
  • bispecific antibody that binds to (human) CCL2 means that the antibody is able to specifically bind to at least two different epitopes on (human) CCL2.
  • bispecific antibody comprises two different antigen binding sites (two different paratopes), each of which is specific for a different epitope of (human) CCL2.
  • the bispecific antibody is capable of binding two different and non overlapping epitopes on CCL2, which means that the two different antigen binding sites do not compete for binding to CCL2.
  • acceptor human framework for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • valent denotes the presence of a specified number of antigen binding sites in an antibody.
  • monovalent binding to an antigen denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antibody.
  • antigen binding site refers to the site or region, i.e. one or several amino acid residues, of an antibody which provides interaction with the antigen.
  • the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs).
  • the antigen binding site of an antibody comprises the comprises amino acid residues from the VH and VL.
  • a native immunoglobulin molecule typically has two antigen binding sites; a Fab molecule typically has a single antigen binding site.
  • Antigen binding moiety refers to a polypeptide molecule comprising an antigen binding site that specifically binds to an antigenic determinant. Antigen binding moieties include antibodies and fragments thereof as further defined herein.
  • antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region.
  • the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art.
  • Useful heavy chain constant regions include any of the five isotypes: a, d, e, g, or m.
  • Useful light chain constant regions include any of the two isotypes: k and l.
  • antigenic determinant refers to a site on a polypeptide macromolecule to which an antigen binding moiety/site binds, forming an antigen binding moiety-antigen complex.
  • useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM).
  • ECM extracellular matrix
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • the bispecific antibodies of the invention are of human IgG isotype, more preferably of humans IgGl isotype.
  • IgG isotype and IgGl isotype refer to the human IgG isotype and human IgGl isotype.
  • the different IgG isotypes exist in the form of slightly different allotypes based on allelic variation among the IgG subclasses ( see Vidarsson et al.; Front Immunol 5 ( 2014) Article 520, 1-17).
  • An "effective amount" of an agent e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Fc domain or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”).
  • a cleaved variant heavy chain also referred to herein as a “cleaved variant heavy chain”.
  • the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present.
  • a heavy chain including a subunit of an Fc domain as specified herein comprised in an antibody or bispecific antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
  • a heavy chain including a subunit of an Fc domain as specified herein, comprised in an antibody or bispecific antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat).
  • Compositions of the invention such as the pharmaceutical compositions described herein, comprise a population of antibodies or bispecific antibodies of the invention.
  • the population of antibodies or bispecific antibodies may comprise molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain.
  • the population of antibodies or bispecific antibodies may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antibodies have a cleaved variant heavy chain.
  • a composition comprising a population of antibodies or bispecific antibodies of the invention comprises an antibody or bispecific antibody comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
  • such a composition comprises a population of antibodies or bispecific antibodies comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat).
  • the lysine at Position 447 numbering according to EU index of Kabat has bee replaced by a glycine (K447G) mutation and the molecules comprise an additional C-terminal glycine-glycine dipeptide (G446 and G447, numbering according to EU index of Kabat).
  • numbering of amino acid residues in the Fc region or constant region is “according to the EU numbering system”, also called “numbering according to the EU index of Kabat” or “Kabat EU numbering”, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).
  • a “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association.
  • a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
  • "Framework” or "FR" refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.
  • the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR-H1(L1)-CDR- H 1 (L 1 )-FR-H2(L2)-CDR-H2(L2)-FR-H3 (L3 )-CDR-H3 (L3 )-FR-H4(L4).
  • full length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Rabat, E.A. et ah, Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3.
  • the subgroup is subgroup kappa I as in Rabat et ak, supra.
  • the subgroup is subgroup III as in Rabat et ak, supra.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • CDRs complementarity determining regions
  • VH VH1, CDR-H2, CDR-H3
  • VL VL1, CDR-L2, CDR-L3
  • Exemplary CDRs herein include:
  • CDR-residues and other residues in the variable domain are numbered herein according to Rabat et al., Rabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding a mono-or bispecific anti-CCL2 antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3).
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (CDRs).
  • FRs conserved framework regions
  • CDRs hypervariable regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et ah, Nature 352 (1991) 624- 628).
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • the invention is based, in part, on the finding that the bispecific antibodies as described herein use different anti-CCL2 antigen binding sites as first and second antigen binding site/moiety.
  • These bispecific anti-CCL2 antibodies bind to certain epitopes of CCL2 with high specificity, and have ability to specifically inhibit binding of CCL2 to its receptor CCR2. They show improved immune complex formation compared to monospecific antibodies and improved CCL2 abrogation in vivo.
  • the specific bispecific anti-CCL2 antibodies in the contorsbody format described herein show in addition valuable properties like low viscosity (which allows e.g. high concentration solutions suitable e.g. for subcutaneous administration)
  • Bispecific antibodies as described herein are monoclonal antibodies that have different binding specificities for at least two different epitopes on CCL2.
  • Multi-specific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No.
  • Engineered antibodies with three or more antigen binding sites including for example, “Octopus antibodies,” or DVD-Ig are also included herein (see, e.g. WO 2001/77342 and WO 2008/024715).
  • Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792, and WO 2013/026831.
  • the bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to CCL2 as well as another different antigen, or two different epitopes of CCL2 (see, e.g., US 2008/0069820 and WO 2015/095539).
  • DAF Double Acting FAb
  • Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CHI/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at ak, MAbs 8 (2016) 1010-20), also called CrossMabs.
  • Asymmetrical binding arms can also be engineered by introducing charged or non- charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.
  • the preferred bispecific antibodies of the present invention are of the following formats:
  • the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain.
  • This basic antibody Fc domain comprising format, the “contorsbody” (CB), is described e.g. in Guy J.Georges et al, Computational and Structural Biotechnology Journal Volume 18, 2020, Pages 1210-1220. See also the scheme in Figure 17, where an example of a bispecific contorsbody format is shown with the different regions and components.
  • the filled circle between the CH3 domains is an optional heterodimerization promoting modification/mutation of the CH3 domains (e.g. knobs into holes, further details described below in the section referring to the heterodimerization promoting FC modifications)
  • polypeptide linker denotes a linker of natural and/or synthetic origin.
  • a polypeptide linker consists of a linear chain of amino acids wherein the 20 naturally occurring amino acids are the monomeric building blocks which are connected by peptide bonds. The chain has a length of from 1 to 15 amino acid residues.
  • the polypeptide linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides.
  • the polypeptide linker has the function to ensure that the antibody domains of the bi specific contorsbody can perform their biological activity by allowing the domains to fold correctly and to be presented properly.
  • the polypeptide linker is a "synthetic peptidic linker" that is designated to be rich in glycine and/or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids.
  • Linker LI and L2 are preferably Glycine-serine linkers; the serine residue is bringing some polarity in the chain to provide solubility to the linker.
  • transition between linker segment and fused protein fragment should preferably not involve a GS motif because there is the potential of a post- translational modification, i.e. O-glycation.
  • the contorsbodies described in this application are then constituted of a terminal glycine. Variations in Length / composition has been tested. Any combination of LI and L2 can be considered. Repetitive glycines are limited to a maximum of 4 consecutive glycines.
  • a linker is made of e.g. of one or two glycines (e.g if C-terminus of the CH3 domains ends with a glycine or is modiefied to end with two glycines) then these one or two glycines have to have to takeninnto account for the limit of a maximum of 4 consecutive glycines.
  • glycines e.g. if C-terminus of the CH3 domains ends with a glycine or is modiefied to end with two glycines
  • these one or two glycines have to have to takeninnto account for the limit of a maximum of 4 consecutive glycines.
  • At the amino- and/or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added.
  • Exemplary linkers with a length of 10 amino acids are e.g.
  • GSGGSGGSGG selected from the group: GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185);GGSGGSGGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), GGGGSGGSGG (SEQ ID NO: 191), preferably GGSGGGGSGG (SEQ ID NO: 188).
  • Analogously further linkers having a length of 5 to 9 or a length of 11 to 15 amino acids can be constructed.
  • the bispecific antibody of the invention comprises an Fc domain composed of a first and a second subunit. It is understood, that the features of the Fc domain described herein in relation to the bispecific antibody can equally apply to an Fc domain comprised in an antibody of the invention.
  • the Fc domain of the bispecific antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule.
  • the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • the two subunits of the Fc domain are capable of stable association with each other.
  • the bispecific antibody of the invention comprises not more than one Fc domain.
  • the Fc domain of the bispecific antibody is an IgG Fc domain.
  • the Fc domain is an IgGi Fc domain.
  • the Fc domain is an IgG4 Fc domain.
  • the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (KabatEU numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).
  • the Fc domain is a human Fc domain.
  • the Fc domain is a human IgGi Fc domain.
  • the Fc domains of IgG isotype are characterized bay various properties based e.g. on their interaction with the Fc gamma Receptors or with the neonatal Fc receptor (FcRn) (see e.g. see Vidarsson et al.; Front Immunol 5 ( 2014) Article 520, 1-17).
  • Bispecific antibodies according to the invention comprise different antigen binding moieties, which may be fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bispecific antibody a modification promoting the association of the desired polypeptides.
  • the Fc domain of the bi specific antibody according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain.
  • the site of most extensive protein- protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.
  • said modification is in the CH3 domain of the Fc domain.
  • the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).
  • These different approaches for improved heavy chain heterodimerization are contemplated as different alternatives in combination with the heavy-light chain modifications (e.g. VH and VL exchange/replacement in one binding arm and the introduction of substitutions of charged amino acids with opposite charges in the CHI/CL interface) in the bispecific antibody which reduce heavy /light chain mispairing and Bence Jones-type side products.
  • said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
  • the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation.
  • Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
  • an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
  • amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the threonine residue at position 366 in (the CH3 domain of) the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V).
  • the threonine residue at position 366 in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Rabat EU index).
  • the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W
  • the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).
  • the antigen binding moiety that binds to the second antigen is fused (optionally via the first antigen binding moiety, which binds to CCL2, and/or a peptide linker) to the first subunit of the Fc domain (comprising the “knob” modification).
  • fusion of the antigen binding moiety that binds a second antigen, such as an activating T cell antigen, to the knob- containing subunit of the Fc domain will (further) minimize the generation of antibodies comprising two antigen binding moieties that bind to an activating T cell antigen (steric clash of two knob-containing polypeptides).
  • CH3 -modification for enforcing the heterodimerization is contemplated as alternatives according to the invention and are described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.
  • the heterodimerization approach described in EP 1870459 is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain.
  • One preferred embodiment for the bispecific antibody of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).
  • the bispecific antibody of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).
  • the bispecific antibody of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said bispecific antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index).
  • the first CH3 domain comprises further amino acid mutation L351K.
  • the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g.
  • T411N, T411R, T411Q, T411K, T411D, T411E or T411W b) D399R, D399W, D399Y or D399K
  • S400E, S400D, S400R, or S400K d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index).
  • a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F.
  • a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F.
  • the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numberings according to Rabat EU index).
  • the heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Rabat EU index).
  • a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A.
  • a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Rabat EU index).
  • the bispecific antibody or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • a first CH3 domain comprises amino acid substitution of R392 or N392 with a negatively charged amino acid (e.g.
  • the first CH3 domain further comprises amino acid substitution of R409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably R409D or R409D).
  • the first CH3 domain further or alternatively comprises amino acid substitution of R439 and/or R370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Rabat EU index).
  • a negatively charged amino acid e.g. glutamic acid (E), or aspartic acid (D)
  • a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Rabat EU index).
  • heterodimerization approach described in WO 2007/110205 can be used alternatively.
  • the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D
  • the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Rabat EU index).
  • wild type (WT) IgG or IgGl refers to a bispecific antibody which comprises an IgG or IgGl constant heavy chain which may comprise the above described modifications/mutations promoting heterodimerization but which does not comprise further Fc domain modifications/mutations increasing or reducing Fc receptor binding and/or effector function as described below.
  • the bispecific anti-CCL2 antibodies were modified using the sweeping technology to enable the bispecific anti-CCL2 antibodies to abrogate free CC12 over longer time periods to enable sustained a biological effect like anti-cancer efficacy in vivo.
  • the present invention provides methods for facilitating antibody mediated antigen uptake into cells, by reducing the antigen-binding activity (binding ability) in the acidic pH range of the above-described antibody to less than its antigen-binding activity in the neutral pH range; and this facilitates antigen uptake into cells.
  • the present invention also provides methods for facilitating antibody-mediated antigen uptake into cells, which are based on altering at least one amino acid in the antigen binding domain of the above-described antibody which facilitates antigen uptake into cells.
  • the present invention also provides methods for facilitating antibody -mediated antigen uptake into cells, which are based on substituting histidine for at least one amino acid or inserting at least one histidine into the antigen-binding domain of the above-described antibody which facilitates antigen uptake into cells.
  • antigen uptake into cells mediated by an antibody means that antigens are taken up into cells by endocytosis.
  • “facilitate the uptake into cells” means that the rate of intracellular uptake of antibody bound to an antigen in plasma is enhanced, and/or the quantity of recycling of uptaken antigen to the plasma is reduced. This means that the rate of uptake into cells is facilitated as compared to the antibody before increasing the human FcRn-binding activity of the antibody in the neutral pH range, or before increasing the human FcRn-binding activity and reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range to less than its antigen-binding activity in the neutral pH range.
  • the rate is improved preferably as compared to intact human IgG, and more preferably as compared to intact human IgG.
  • whether antigen uptake into cells is facilitated by an antibody can be assessed based on an increase in the rate of antigen uptake into cells.
  • the rate of antigen uptake into cells can be calculated, for example, by monitoring over time reduction in the antigen concentration in the culture medium containing human FcRn-expressing cells after adding the antigen and antibody to the medium, or monitoring over time the amount of antigen uptake into human FcRn-expressing cells.
  • the rate of antigen elimination from the plasma can be enhanced by administering antibodies.
  • whether antibody-mediated antigen uptake into cells is facilitated can also be assessed, for example, by testing whether the rate of antigen elimination from the plasma is accelerated or whether the total antigen concentration in plasma is reduced by administering an antibody.
  • total antigen concentration in plasma means the sum of antibody bound antigen and non-bound antigen concentration, or "free antigen concentration in plasma” which is antibody non-bound antigen concentration.
  • Various methods to measure “total antigen concentration in plasma” or “free antigen concentration in plasma” is well known in the art as described hereinafter.
  • “Intact human IgG” (or “wild type (WT) human IgG) as used herein is meant an unmodified (except with respect to the potential modifications for heterodimerization above) human IgG and is not limited to a specific class of IgG .
  • human IgGl, IgG2, IgG3 or IgG4 can be used as "intact human IgG” as long as it can bind to the human FcRn in the acidic pH range.
  • "intact human IgG” can be human IgGl .
  • the present invention also provides methods for increasing the number of antigens to which a single antibody can bind. More specifically, the present invention provides methods for increasing the number of antigens to which a single antibody having human FcRn-binding activity in the acidic pH range can bind, by increasing the human FcRn-binding activity of the antibody in the neutral pH range. The present invention also provides methods for increasing the number of antigens to which a single antibody having human FcRn-binding activity in the acidic pH range can bind, by altering at least one amino acid in the human FcRn-binding domain of the antibody.
  • the present invention provides methods for facilitating antibody-mediated antigen uptake into cells. More specifically, the present invention provides methods for facilitating the antigen uptake into cells by an antibody having human FcRn-binding activity in the acidic pH range, which are based on increasing the human FcRn- binding activity of the antibody in the neutral pH range. The present invention also provides methods for improving antigen uptake into cells by an antibody having human FcRn-binding activity in the acidic pH range, which are based on altering at least one amino acid in the human FcRn-binding domain of the antibody.
  • the present invention also provides methods for facilitating antigen uptake into cells by an antibody having human FcRn-binding activity in the acidic pH range, which are based on using a human FcRn-binding domain comprising an amino acid sequence with a substitution of a different amino acid for at least one amino acid selected from those of positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the parent IgG Fc domain of the human FcRn-binding domain comprising the Fc domain of parent IgG.
  • a human FcRn-binding domain comprising an amino acid sequence with a substitution
  • the present invention also provides methods for facilitating antibody-mediated antigen uptake into cells, by reducing the antigen-binding activity (binding ability) in the acidic pH range of the above-described antibody to less than its antigen binding activity in the neutral pH range; and this facilitates antigen uptake into cells.
  • the present invention also provides methods for facilitating antibody-mediated antigen uptake into cells, which are based on altering at least one amino acid in the antigen-binding domain of the above-described antibody which facilitates antigen uptake into cells.
  • the present invention also provides methods for facilitating antibody-mediated antigen uptake into cells, which are based on substituting histidine for at least one amino acid or inserting at least one histidine into the antigen binding domain of the above-described antibody which facilitates antigen uptake into cells.
  • antigen uptake into cells mediated by an antibody means that antigens are taken up into cells by endocytosis.
  • “facilitate the uptake into cells” means that the rate of intracellular uptake of antibody bound to an antigen in plasma is enhanced, and/or the quantity of recycling of uptaken antigen to the plasma is reduced. This means that the rate of uptake into cells is facilitated as compared to the antibody before increasing the human FcRn-binding activity of the antibody in the neutral pH range, or before increasing the human FcRn-binding activity and reducing the antigen-binding activity (binding ability) of the antibody in the acidic pH range to less than its antigen-binding activity in the neutral pH range.
  • the rate is improved preferably as compared to intact human IgG, and more preferably as compared to intact human IgG.
  • whether antigen uptake into cells is facilitated by an antibody can be assessed based on an increase in the rate of antigen uptake into cells.
  • the rate of antigen uptake into cells can be calculated, for example, by monitoring over time reduction in the antigen concentration in the culture medium containing human FcRn-expressing cells after adding the antigen and antibody to the medium, or monitoring over time the amount of antigen uptake into human FcRn-expressing cells.
  • the rate of antigen elimination from the plasma can be enhanced by administering antibodies.
  • whether antibody-mediated antigen uptake into cells is facilitated can also be assessed, for example, by testing whether the rate of antigen elimination from the plasma is accelerated or whether the total antigen concentration in plasma is reduced by administering an antibody.
  • total antigen concentration in plasma means the sum of antibody bound antigen and non-bound antigen concentration, or "free antigen concentration in plasma” which is antibody non-bound antigen concentration.
  • Various methods to measure “total antigen concentration in plasma” or “free antigen concentration in plasma” is well known in the art as described hereinafter.
  • “Intact human IgG” (or “wild type IgG”) as used herein is meant an unmodified human IgG ((except with respect to the potential modifications for heterodimerization above) and is not limited to a specific class of IgG.
  • human IgGl, IgG2, IgG3 or IgG4 can be used as "intact human IgG” as long as it can bind to the human FcRn in the acidic pH range.
  • "intact human IgG” can be human IgGl .
  • Parent IgG as used herein means an unmodified IgG that is subsequently modified to generate a variant as long as a modified variant of parent IgG can bind to human FcRn in the acidic pH range (therefore, parent IgG does not necessary requires binding activity to human FcRn in the acidic condition).
  • the parent IgG may be a naturally occurring IgG, or a variant or engineered version of a naturally occurring IgG.
  • Parent IgG may refer to the polypeptide itself, compositions that comprise the parent IgG, or the amino acid sequence that encodes it. It should be noted that "parent IgG” includes known commercial, recombinantly produced IgG as outlined below.
  • parent IgG is not limited and may be obtained from any organisms of non-human animals or human.
  • organism is selected from mouse, rat, guinea pig, hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, and non human primate.
  • parent IgG can also be obtained from cynomolgus, marmoset, rhesus, chimpanzee or human.
  • "parent IgG” is obtained from human IgGl but not limited to a specific class of IgG. This means that human IgGl, IgG2, IgG3, or IgG4 can be appropriately used as "parent IgG".
  • any class or subclass of IgGs from any organisms hereinbefore can be preferably used as "parent IgG".
  • Example of variant or engineered version of a naturally occurring IgG is described in Curr Opin Biotechnol. 2009 Dec; 20(6): 685- 91, Curr Opin Immunol. 2008 Aug; 20(4): 460-70, Protein Eng Des Sel. 2010 Apr; 23(4): 195-202, WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, but not limited thereto.
  • the present invention also provides methods for increasing the ability to eliminate plasma antigen by administering antibodies.
  • "methods for increasing the ability to eliminate plasma antigen” is synonymous to "methods for augmenting the ability of an antibody to eliminate antigen from plasma”. More specifically, the present invention provides methods for increasing the ability to eliminate plasma antigen by an antibody having human FcRn-binding activity in the acidic pH range, by increasing the human FcRn-binding activity of the antibody in the neutral pH range. The present invention also provides methods for increasing the ability to eliminate plasma antigen by an antibody having human FcRn-binding activity in the acidic pH range, which are based on altering at least one amino acid in the human FcRn-binding domain of the antibody.
  • the present invention also provides methods for increasing the ability to eliminate plasma antigen by an antibody having human FcRn-binding activity in the acidic pH range, by using a human FcRn-binding domain comprising an amino acid sequence with a substitution of at least one amino acid selected from those of positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the parent IgG Fc domain of the human FcRn-binding domain comprising the Fc domain of parent IgG with a different amino acid.
  • a human FcRn-binding domain comprising an amino acid sequence with a substitution of at least one
  • the present invention also provides methods for increasing the ability to eliminate plasma antigen by an antibody, by reducing the antigen-binding activity in the acidic pH range of the above-described antibody with improved ability to eliminate plasma antigen as compared to the antigen-binding activity in the neutral pH range.
  • the present invention also provides methods for increasing the ability to eliminate plasma antigen by an antibody, by altering at least one amino acid in the antigen binding domain of the above-described antibody with improved ability to eliminate plasma antigen.
  • the present invention also provides methods for increasing the ability to eliminate plasma antigen by administering an antibody, by substituting histidine for at least one amino acid or inserting at least one histidine into the antigen binding domain of the above-described antibody with improved ability to eliminate plasma antigen.
  • the "ability to eliminate plasma antigen” means the ability to eliminate antigen from the plasma when antibodies are administered or secreted in vivo.
  • “increase in the ability of antibody to eliminate plasma antigen” herein means that the rate of antigen elimination from the plasma is accelerated upon administration of the antibody as compared to before increasing the human FcRn-binding activity of the antibody in the neutral pH range or before increasing the human FcRn-binding activity and simultaneously reducing its antigen-binding activity in the acidic pH range to less than that in the neutral pH range.
  • Increase in the activity of an antibody to eliminate antigen from the plasma can be assessed, for example, by administering a soluble antigen and an antibody in vivo, and measuring the concentration of the soluble antigen in plasma after administration.
  • a form of soluble antigen can be antibody bound antigen or antibody non bound antigen whose concentration can be determined as "antibody bound antigen concentration in plasma” and “antibody non-bound antigen concentration in plasma” respectively (The latter is synonymous to "free antigen concentration in plasma”.
  • total antigen concentration in plasma means the sum of antibody bound antigen and non-bound antigen concentration, or "free antigen concentration in plasma” which is antibody non-bound antigen concentration, the concentration of soluble antigen can be determined as “total antigen concentration in plasma”.
  • Various methods for measuring "total antigen concentration in plasma” or “free antigen concentration in plasma” are well known in the art as described hereinafter.
  • the present invention also provides methods for improving the pharmacokinetics of antibodies. More specifically, the present invention provides methods for improving the pharmacokinetics of the antibody having human FcRn-binding activity in the acidic pH range by increasing the human FcRn-binding activity of the antibody in the neutral pH range. Furthermore, the present invention provides methods for improving the pharmacokinetics of an antibody having human FcRn-binding activity in the acidic pH range by altering at least one amino acid in the human FcRn-binding domain of the antibody.
  • the present invention also provides methods for improving the pharmacokinetics of an antibody having human FcRn-binding activity in the acidic pH range by using a human FcRn-binding domain comprising an amino acid sequence with a substitution of different amino acid for at least one amino acid selected from those of positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the parent IgG Fc domain of the human FcRn-binding domain comprising the Fc domain of IgG.
  • a human FcRn-binding domain comprising an amino acid sequence with a substitution of different amino acid for at least one amino
  • the plasma concentration of free antigen not bound to the antibody or the ratio of free antigen concentration to the total concentration can be determined by methods known to those skilled in the art, for example, by the method described in Pharm Res. 2006 Jan; 23 (1): 95-103.
  • whether the antigen is bound to an antibody that neutralizes the antigen function can be assessed by testing whether the antigen function is neutralized. Whether the antigen function is neutralized can be assessed by assaying an in vivo marker that reflects the antigen function. Whether the antigen is bound to an antibody that activates the antigen function (agonistic molecule) can be assessed by assaying an in vivo marker that reflects the antigen function.
  • the assays are preferably carried out after a certain period of time has passed after administration of the antibody.
  • the period after administration of the antibody is not particularly limited; those skilled in the art can determine the appropriate period depending on the properties and the like of the administered antibody. Such periods include, for example, one day after administration of the antibody, three days after administration of the antibody, seven days after administration of the antibody, 14 days after administration of the antibody, and 28 days after administration of the antibody.
  • plasma antigen concentration means either "total antigen concentration in plasma” which is the sum of antibody bound antigen and non-bound antigen concentration or "free antigen concentration in plasma” which is antibody non-bound antigen concentration.
  • Total antigen concentration in plasma can be lowered by administration of antibody of the present invention by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200- fold, 500-fold, 1,000-fold, or even higher compared to the administration of a reference antibody comprising the intact human IgG Fc domain as a human FcRn- binding domain or compared to when antigen-binding domain molecule of the present invention is not administered.
  • the invention provides bispecific anti-CCL2 antibodies that exhibit pH-dependent binding characteristics.
  • pH-dependent binding means that the antibody exhibits "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH” (for purposes of the present disclosure, both expressions may be used interchangeably).
  • antibodies "with pH- dependent binding characteristics” include antibodies that bind to CCL2 with higher affinity at neutral pH than at acidic pH.
  • the bispecific antibodies of the present invention bind to CCL2 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times higher affinity at neutral pH than at acidic pH.
  • the antibodies bind to CCL2 with higher affinity at pH7.4 than at pH5.8.
  • the antibodies bind to CCL2 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times higher affinity at pH7.4 than at pH5.8.
  • an antigen is a soluble protein
  • the binding of an antibody to the antigen can result in an extended half-life of the antigen in plasma (i.e., reduced clearance of the antigen from plasma), since the antibody can have a longer half-life in plasma than the antigen itself and may serve as a carrier for the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the endosomal pathway in cell (Roopenian, Nat. Rev. Immunol. 7(9): 715-725 (2007)).
  • an antibody with pH-dependent binding characteristics which binds to its antigen in neutral extracellular environment while releasing the antigen into acidic endosomal compartments following its entry into cells, is expected to have superior properties in terms of antigen neutralization and clearance relative to its counterpart that binds in a pH-independent manner (Igawa et al., Nature Biotechnol. 28(11): 1203-1207 (2010); Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009/125825).
  • the “affinity” of an antibody for CCL2, for purposes of the present disclosure is expressed in terms of the KD of the antibody.
  • the KD of an antibody refers to the equilibrium dissociation constant of an antibody-antigen interaction. The greater the KD value is for an antibody binding to its antigen, the weaker its binding affinity is for that particular antigen. Accordingly, as used herein, the expression “higher affinity at neutral pH than at acidic pH” (or the equivalent expression "pH-dependent binding") means that the KD of the antibody binding to CCL2 at acidic pH is greater than the KD of the antibody binding to CCL2 at neutral pH.
  • an antibody is considered to bind to CCL2 with higher affinity at neutral pH than at acidic pH if the KD of the antibody binding to CCL2 at acidic pH is at least 2 times greater than the KD of the antibody binding to CCL2 at neutral pH.
  • the present invention includes antibodies that bind to CCL2 at acidic pH with a KD that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody binding to CCL2 at neutral pH.
  • the KD value of the antibody at neutral pH can be 10-7 M, 10-8 M, 10- 9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • the KD value of the antibody at acidic pH can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or greater.
  • an antibody is considered to bind to with a higher affinity at neutral pH than at acidic pH if the KD of the antibody binding to CCL2 at pH5.8 is at least 2 times greater than the KD of the antibody binding to CCL2 at pH7.4.
  • the provided antibodies bind to CCL2 at pH5.8 with a KD that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the KD of the antibody binding to CCL2 at pH7.4.
  • the KD value of the antibody at pH7.4 can be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • the KD value of the antibody at pH5.8 can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or greater.
  • the binding properties of an antibody for a particular antigen may also be expressed in terms of the kd of the antibody.
  • the kd of an antibody refers to the dissociation rate constant of the antibody with respect to a particular antigen and is expressed in terms of reciprocal seconds (i.e., sec-1).
  • An increase in kd value signifies weaker binding of an antibody to its antigen.
  • the present invention therefore includes antibodies that bind to CCL2 with a higher kd value at acidic pH than at neutral pH.
  • the present invention includes antibodies that bind to CCL2 at acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody binding to CCL2 at neutral pH.
  • the kd value of the antibody at neutral pH can be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s, or less.
  • the kd value of the antibody at acidic pH can be 10-3 1/s, 10-2 1/s, 10- 1 1/s, or greater.
  • the invention also includes antibodies that bind to CCL2 with a higher kd value at pH5.8 than at pH7.4.
  • the invention includes antibodies that bind to CCL2 at pH5.8 with a kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater than the kd of the antibody binding to CCL2 at pH7.4.
  • the kd value of the antibody at pH7.4 can be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s, or less.
  • the kd value of the antibody at pH5.8 can be 10-3 1/s, 10-2 1/s, 10-1 1/s, or greater.
  • a "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH” is expressed in terms of the ratio of the KD value of the antibody binding to CCL2 at acidic pH to the KD value of the antibody binding to CCL2 at neutral pH (or vice versa).
  • an antibody may be regarded as exhibiting "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH", for purposes of the present invention, if the antibody exhibits an acidic/neutral KD ratio of 2 or greater.
  • the pH5.8/pH7.4 KD ratio for an anti-CCL2 antibody of the present invention is 2 or greater.
  • the acidic/neutral KD ratio for an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater.
  • the KD value of the antibody at neutral pH can be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • the KD value of the antibody at acidic pH can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or greater.
  • an antibody may be regarded as exhibiting "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH", if the antibody exhibits an pH5.8/pH7.4 KD ratio of 2 or greater.
  • the pH5.8/pH7.4 KD ratio for the antibody can be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater.
  • the KD value of the antibody at pH7.4 can be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • the KD value of the antibody at pH5.8 can be 10- 9 M, 10-8 M, 10-7 M, 10-6 M, or greater.
  • a "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH” is expressed in terms of the ratio of the kd value of the antibody binding to CCL2 at acidic pH to the kd value of the antibody binding to CCL2 at neutral pH (or vice versa).
  • an antibody may be regarded as exhibiting "reduced binding to CCL2 at acidic pH as compared to its binding at neutral pH", for purposes of the present invention, if the antibody exhibits an acidic/neutral kd ratio of 2 or greater.
  • the pH5.8/pH7.4 kd ratio for an antibody of the present invention is 2 or greater.
  • the acidic/neutral kd ratio for an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater.
  • the kd value of the antibody at neutral pH can be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s, or less.
  • the kd value of the antibody at acidic pH can be 10-3 1/s, 10-2 1/s, 10-1 1/s, or greater.
  • the pH5.8/pH7.4 kd ratio for an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or greater.
  • the kd value of the antibody at pH7.4 can be 10-2 1/s, 10-3 1/s, 10-4 1/s, 10-5 1/s, 10-6 1/s, or less.
  • the kd value of the antibody at pH5.8 can be 10-3 1/s, 10-2 1/s, 10-1 1/s, or greater.
  • the expression "acidic pH” means a pH of 4.0 to 6.5.
  • acidic pH includes pH values of any one of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5.
  • the "acidic pH” is 5.8.
  • neutral pH means a pH of 6.7 to about 10.0.
  • the expression “neutral pH” includes pH values of any one of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.
  • the "neutral pH” is 7.4.
  • KD values, and kd values may be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. KD values, and kd values can be determined at 25 degrees C or 37 degrees C.
  • the invention provides a bispecific anti-CCL2 antibody that forms an immune complex (i.e. antigen-antibody complex) with CCL2.
  • an immune complex i.e. antigen-antibody complex
  • two or more bispecific anti-CCL2 antibodies bind to two or more CCL2 molecules to form an immune complex. This is possible because CCL2 exists as a homodimer containing two CCL2 molecules while an antibody has two antigen binding sites.
  • the resulting immune complex can strongly bind to Fc receptors existing on cell surfaces due to avidity effects through the Fc regions of the antibodies in the complex and can then be taken up into the cell with high efficiency.
  • the above-mentioned anti-CCL2 antibody capable of forming an immune complex containing two or more anti-CCL2 antibodies and two or more CCL2 molecules can lead to a rapid clearance of CCL2 from plasma in a living body, via the strong binding to Fc receptors due to avidity effects.
  • an antibody with pH-dependent binding characteristics is thought to have superior properties in terms of antigen neutralization and clearance relative to its counterpart that binds in a pH-independent manner (Igawa et ak, Nature Biotech. 28(11): 1203-1207 (2010); Devanaboyina et al. mAbs 5(6):851-859 (2013); WO 2009/125825). Therefore, an antibody having both properties above, that is, an antibody which has pH-dependent binding characteristics and which forms an immune complex containing two or more antibodies with two or more antigens, is expected to have even more superior properties for highly accelerated elimination of antigens from plasma (WO 2013/081143).
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity comprising at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, according to EU numbering.
  • the invention provides polypeptides comprising a variant Fc region with enhanced FcgammaRIIb-binding activity comprising an amino acid alteration at position 236 according to EU numbering.
  • the invention provides polypeptides comprising a variant Fc region with enhanced FcgammaRIIb-binding activity comprising at least two amino acid alterations comprising: (a) one amino acid alteration at position 236, and (b) at least one amino acid alteration of at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering.
  • the variant Fc region comprises an amino acid alteration of at least one position selected from the group consisting of: 231, 232, 235, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering.
  • the variant Fc region comprises an amino acid alteration of at least one position selected from the group consisting of: 268, 295, 326, and 330, according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity comprising amino acid alterations of any one of the following (l)-(37): (1) positions 231, 236, 239, 268 and 330; (2) positions 231, 236, 239, 268, 295 and 330; (3) positions 231, 236, 268 and 330; (4) positions 231, 236, 268, 295 and 330; (5) positions 232, 236, 239, 268, 295 and 330; (6) positions 232, 236, 268, 295 and 330; (7) positions 232, 236, 268 and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) positions 235, 236, 268, 295 and 330; (10) positions 235, 236, 268 and 330; (11) positions 235, 236, 268, 330 and 396; (12) positions 235, 236, 268 and 396; (13) positions 236, 239, 268, 295, 298 and
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises at least one amino acid selected from the group consisting of: (a) Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 231; (b) Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, Tyr at position 232; (c) Asp at position 233; (d) Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu, His, He, Leu, Met, Asn, Gin, Ser, Thr, Val at position 236; (g) Asp, Tyr at position 237; (h) Glu, He, Met, Gin, Tyr at position 238; (i) He, Leu, Asn
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises at least one amino acid alteration (e.g., substitution) selected from the group consisting of: (a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp at position 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f) Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position 298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys, Met at position 396; according to EU numbering.
  • amino acid alteration e.g., substitution
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asn at position 236, Glu at position 268, Lys at position 330, and Met at position 396; according to EU numbering.
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asn at position 236, Asp at position 268, and Lys at position 330; according to EU numbering.
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asn at position 236, Asp at position 268, Leu at position 295, and Lys at position 330; according to EU numbering.
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Thr at position 236, Asp at position 268, and Lys at position 330; according to EU numbering.
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330; according to EU numbering.
  • the variant Fc region with enhanced FcgammaRIIb-binding activity comprises amino acid alterations (e.g., substitutions) of: Trp at position 235, Asn at position 236, Asp at position 268, Leu at position 295, Thr at position 326, and Lys at position 330; according to EU numbering.
  • the invention provides isolated polypeptides comprising variant Fc regions with increased isoelectric point (pi).
  • a variant Fc region described herein comprises at least two amino acid alterations in a parent Fc region.
  • each of the amino acid alterations increases the isoelectric point (pi) of the variant Fc region compared with that of the parent Fc region.
  • pi may be either a theoretical or an experimentally determined pi.
  • the value of pi can be determined, for example, by isoelectric focusing known to those skilled in the art.
  • the value of a theoretical pi can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.).
  • the pi value may be increased, for example, at least by 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, at least by 0.6, 0.7, 0.8, 0.9, or more, at least by 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or more, or at least by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0 or more, as compared to before modification.
  • the amino acid for increased pi can be exposed on the surface of the variant Fc region.
  • an amino acid that can be exposed on the surface generally refers to an amino acid residue located on the surface of a polypeptide constituting a variant Fc region.
  • An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can be in contact with solvent molecules (which in general are mostly water molecules).
  • solvent molecules which in general are mostly water molecules.
  • the side chain does not necessarily have to be wholly in contact with solvent molecules, and when even a portion of the side chain is in contact with the solvent molecules, the amino acid is defined as an "amino acid residue located on the surface".
  • the amino acid residues located on the surface of a polypeptide also include amino acid residues located close to the surface and thereby can have an electric charge influence from another amino acid residue whose side chain, even partly, is in contact with the solvent molecules.
  • Those skilled in the art can prepare a homology model of a polypeptide for example, using commercially available softwares. Alternatively, it is possible to use methods known to those skilled in the art, such as X-ray crystallography.
  • the amino acid residues that can be exposed on the surface are determined, for example, using coordinates from a three- dimensional model using a computer program such as Insightll program (Accelrys). Surface-exposable sites may be determined using algorithms known in the technical field (for example, Lee and Richards (J. Mol. Biol.
  • a polypeptide comprises both the variant Fc region and an antigen-binding domain.
  • the antigen is a soluble antigen.
  • the antigen is present in biological fluids (for example, plasma, interstitial fluid, lymphatic fluid, ascitic fluid, and pleural fluid) of subjects.
  • the antigen may also be a membrane antigen.
  • antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions.
  • ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration.
  • metal ions refer to ions of group I elements except hydrogen, such as alkaline metals and the copper group elements, group II elements such as alkaline earth metals and zinc group elements, group III elements except boron, group IV elements except carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium.
  • metal ions include, for example, calcium ion, as described in WO 2012/073992 and WO 2013/125667.
  • ion concentration condition may be a condition that focuses on differences in the biological behavior of an antigen-binding domain between a low ion concentration and a high ion concentration.
  • antigen-binding activity of an antigen-binding domain changes according to ion concentration conditions means that the antigen-binding activity of an antigen-binding domain changes between a low ion concentration and a high ion concentration (such an antigen-binding domain is referred to herein as "ion concentration-dependent antigen-binding domain").
  • the antigen-binding activity of an antigen-binding domain under a high ion concentration condition may be higher (stronger) or lower (weaker) than that under a low ion concentration condition.
  • ion concentration-dependent antigen-binding domains such as pH-dependent antigen-binding domains or calcium ion concentration-dependent antigen-binding domains
  • WO 2009/125825, WO 2012/073992, and WO 2013/046722 can be obtained by known methods, for example, described in WO 2009/125825, WO 2012/073992, and WO 2013/046722.
  • the antigen-binding activity of an antigen-binding domain under a high calcium ion concentration condition may be higher than under a low calcium ion concentration condition.
  • the high calcium ion concentration is not particularly limited to but may be a concentration selected between 100 micro M and 10 mM, between 200 micro M and 5 mM, between 400 micro M and 3 mM, between 200 micro M and 2 mM, between 400 micro M and 1 mM, or between 500 micro M and 2.5 mM, which is preferable to be close to the plasma (blood) concentration of calcium ion in vivo.
  • the low calcium ion concentration is not particularly limited to but may be a concentration selected between 0.1 micro M and 30 micro M, between 0.2 micro M and 20 micro M, between 0.5 micro M and 10 micro M, between 1 micro M and 5 micro M, or between 2 micro M and 4 micro M, which is preferable to be close to the concentration of calcium ion in early endosomes in vivo.
  • the ratio between the antigen-binding activities under a low calcium ion concentration condition and a high calcium ion concentration condition is not limited but the ratio of the dissociation constant (KD) under a low calcium ion concentration condition to the KD under a high calcium ion concentration condition, i.e., KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition), is 2 or more, 10 or more, or 40 or more.
  • the upper limit of the ratio may be 400, 1000, or 10000, as long as such an antigen-binding domain can be produced by techniques known to those skilled in the art.
  • the dissociation rate constant (kd) can be used instead of the KD.
  • the ratio of the kd under a low calcium ion concentration condition to the kd under a high calcium ion concentration condition is 2 or more, 5 or more, 10 or more, or 30 or more.
  • the upper limit of the ratio may be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the common technical knowledge of those skilled in the art.
  • the antigen-binding activity of an antigen-binding domain under a low hydrogen ion concentration may be higher than under a high hydrogen ion concentration (acidic pH).
  • the acidic pH may be, for example, a pH selected from pH4.0 to pH6.5, selected from pH4.5 to pH6.5, selected from pH5.0 to pH6.5, or selected from pH5.5 to pH6.5, which is preferable to be close to the in vivo pH in early endosomes.
  • the acidic pH may also be, for example, pH5.8 or pH6.0. In particular embodiments, the acidic pH is pH5.8.
  • the neutral pH may be, for example, a pH selected from pH6.7 to pHlO.O, selected from pH6.7 to pH9.5, selected from pH7.0 to pH9.0, or selected from pH7.0 to pH8.0, which is preferable to be close to the in vivo pH in plasma (blood).
  • the neutral pH may also be, for example, pH7.4 or pH7.0.
  • the neutral pH is pH7.4.
  • the ratio between the antigen-binding activities under an acidic pH condition and a neutral pH condition is not limited but the ratio of the dissociation constant (KD) under an acidic pH condition to the KD under a neutral pH condition, i.e., KD (acidic pH condition)/KD (neutral pH condition), is 2 or more, 10 or more, or 40 or more.
  • the upper limit of the ratio may be 400, 1000, or 10000, as long as such an antigen-binding domain can be produced by techniques known to those skilled in the art.
  • the dissociation rate constant (kd) can be used instead of the KD.
  • the ratio of the kd under an acidic pH condition to the kd under a neutral pH condition i.e., kd (acidic pH condi tion)/kd (neutral pH condition) is 2 or more, 5 or more, 10 or more, or 30 or more.
  • the upper limit of the ratio may be 50, 100, or 200, as long as the antigen-binding domain can be produced based on the common technical knowledge of those skilled in the art.
  • At least one amino acid residue is substituted with an amino acid residue with a side-chain pKa of 4.0-8.0, and/or at least one amino acid with a side-chain pKa of 4.0-8.0 is inserted in the antigen-binding domain, as described in WO 2009/125825.
  • the amino acid may be substituted and/or inserted at any site as long as the antigen-binding activity of the antigen-binding domain becomes weaker under an acidic pH condition than under a neutral pH condition as compared to before the substitution or insertion.
  • the site may be within the variable region or CDR.
  • amino acids that are substituted or inserted can be appropriately determined by those skilled in the art; and the number may be one or more.
  • Amino acids with a side-chain pKa of 4.0-8.0 can be used to change the antigen-binding activity of the antigen-binding domain according to the hydrogen ion concentration condition.
  • Such amino acids include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US2009/0035836), m-N02-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et ah, Bioorg. Med. Chem. 11(17):3761-3768 (2003)).
  • Amino acids with a side-chain pKa of 6.0-7.0 can also be used, which include, e.g., His (H).
  • preferable antigen-binding domains for the variant Fc region with increased pi are described and can be obtained by methods described in WO2016/125495 and WO2017/046994.
  • the variant Fc region with increased pi comprises at least two amino acid alterations of at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EU numbering.
  • the variant Fc region with increased pi comprises at least two amino acid alterations of at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with increased pi comprising amino acid alterations of any one of the following (1)- (10): (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413; according to EU numbering.
  • (1)- (10) (1) positions 311 and 341; (2) positions 311 and 343; (3) positions 311, 343 and 413; (4) positions 311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401; (7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401 and 413; and (10) positions 402 and 413; according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity and increased pi comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration of at least one position selected from the group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332, 334, and 396, according to EU numbering, and (b) at least two amino acid alterations of at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity and increased pi, and that comprise at least three amino acid alterations comprising: (a) at least one amino acid alteration of at least one position selected from the group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering, and (b) at least two amino acid alterations of at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity and increased pi comprising amino acid alterations of any one of the following (l)-(9): (1) positions 235, 236, 268, 295, 311, 326, 330 and 343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions 236, 268, 295, 311, 330 and 413; (4) positions 236, 268, 311, 330, 396 and 399; (5) positions 236, 268, 311, 330 and 343; (6) positions 236, 268, 311, 330, 343 and 413; (7) positions 236, 268, 311, 330, 384 and 413; (8) positions 236, 268, 311, 330 and 413; and (9) positions 236, 268, 330, 396, 400 and 413; according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity and increased pi comprising at least three amino acid alterations comprising: (a) at least one amino acid alteration of at least one position selected from the group consisting of: 234, 238, 250, 264, 267, 307, and 330, and (b) at least two amino acid alterations of at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EU numbering.
  • the polypeptides comprise at least two amino acid alterations of at least two positions selected from the group consisting of: 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EU numbering.
  • the invention provides polypeptides comprising variant Fc regions with enhanced FcgammaRIIb-binding activity and increased pi comprising amino acid alterations of any one of the following (1)-(16): (1) positions 234, 238, 250, 264, 307, 311, 330 and 343; (2) positions 234, 238, 250, 264, 307, 311, 330 and 413; (3) positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) positions 234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) positions 234, 238, 250, 267, 307, 311, 330 and 343; (6) positions 234, 238, 250, 267, 307, 311, 330 and 413; (7)
  • amino acid alterations performed for other purpose(s) can be combined in a variant Fc region described herein.
  • amino acid substitutions that improve FcRn-binding activity Hinton et al., J. Immunol. 176(1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Petkova et al., Inti. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol.
  • polypeptides with the property of promoting antigen clearance which are described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides with the property of specific binding to a target tissue, which are described in WO 2013/180200, polypeptides with the property for repeated binding to a plurality of antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with a variant Fc region described herein.
  • amino acid alterations disclosed in EP1752471 and EP1772465 may be combined in CH3 of a variant Fc region described herein.
  • amino acid alterations that decrease the pi of the constant region (WO 2012/016227) may be combined in a variant Fc region described herein.
  • amino acid alterations that increase the pi of the constant region (WO 2014/145159) may be combined in a variant Fc region described herein.
  • amino acid alterations that increase the pi of the constant region may be combined in a variant Fc region described herein.
  • alteration may include, for example, substitution at al least one position selected from the group consisting of 311, 343, 384, 399, 400, and 413 according to EU numbering.
  • substitution may be a replacement of an amino acid with Lys or Arg at each position.
  • Amino acid alterations of enhancing human FcRn-binding activity under acidic pH can also be combined in a variant Fc region described herein.
  • such alterations may include, for example, substitution of Leu for Met at position 428 and substitution of Ser for Asn at position 434, according to EU numbering (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)); substitution of Ala for Asn at position 434 (Deng et al., Metab. Dispos. 38(4):600-605 (2010)); substitution of Tyr for Met at position 252, substitution of Thr for Ser at position 254 and substitution of Glu for Thr at position 256 (Dall'Acqua et al., J. Biol. Chem.
  • Such alterations may include, for example, at least one alteration selected from the group consisting of substitution of Leu for Met at position 428, substitution of Ala for Asn at position 434 and substitution of Thr for Tyr at position 436. Those alterations may further include substitution of Arg for Gin at position 438 and/or substitution of Glu for Ser at position 440 (WO2016/125495).
  • One embodiment of the invention is a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2 and a second different antigen-binding site that (specifically) binds to a second different epitope on human CCL2, wherein the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93; O) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.
  • One embodiment of the invention is a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2 and a second different antigen-binding site that (specifically) binds to a second different epitope on human CCL2, wherein the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids), Hinge is a heavy chain hinge region,
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • said first antigen-binding site comprises a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a CDR-H2 comprising the amino acid sequence GX 1 IX 2 IFX 3 TANYAQKFQG of SEQ ID NO: 58 wherein X 1 is V, X 2 is P, and X 3 is H, and (c) a CDR- H3 comprising the amino acid sequence YDAHYGELDF of SEQ ID NO: 59; and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2 comprising the amino acid sequence DASDRAE of SEQ ID NO: 61, and (f) a CDR-L3 comprising the amino acid sequence HQYIHLHSFT of
  • One embodiment of the invention is a bispecific antibody comprising a first antigen binding site that (specifically) binds to a first epitope on human CCL2 and a second different antigen-binding site that (specifically) binds to a second different epitope on human CCL2, wherein the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain, wherein i) the VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93.
  • LI and L2 are polypeptide linkers comprising the amino acids glycine and serine whrein repetitive glycines are limited to a maximum of 4 consecutive glycines and no serine is directly connected to another serine.
  • LI is a polypeptide linker with a length of 9 to 11 amino acids
  • L2 is a polypeptide linker with a length of 9 to 11 amino acids
  • LI and L2 are polypeptide linkers selected from the group of : GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185);GGSGGSGGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), and GGGGSGGSGG (SEQ ID NO: 191.
  • LI is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188), and L2 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188).
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgG isotype, preferably of human IgGl isotype.
  • the bispecific antibody described herein is not cross-reactive to other human CCL homologs in particular it shows 100 time less binding to other CCL homologs (selected from the group of CCL8, CCL7, and CCL 13) compared to the binding to CCL2
  • the bispecific antibody described herein binds to the first and second epitope on human CCL2 in ion-dependent manner.
  • the bispecific antibody described herein binds to human CCL2 in pH dependent manner and wherein the first antigen binding site and the second antigen binding site both bind to CCL2 with a higher affinity at neutral pH than at acidic pH.
  • the bispecific antibody described herein binds to human CCL2 with a 10 times higher affinity at pH 7.4, than at pH 5.8
  • the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of the bispecific antibody comprising a constant heavy chain domain of human wild type IgGl isotype (or the Fc domain thereof ) is at least 15 fold higher, in particular at least 20 fold higher, compared to the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody comprising a Fc gamma receptor silenced constant heavy chain domain of human IgGl isotype (or the Fc domain thereof) comprising the mutations L234A, L235A, P329G (Kabat EU numbering), when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and O.lmg/kg human CCL2 was administered at a single dose of 10 ml/kg into FcRn transgenic mice.
  • the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of the bispecific antibody comprising a constant heavy chain domain of human wild type IgGl isotype (or the Fc domain thereof) is at least two fold higher (in one embodiment at least 5 fold higher, in one embodiment at least 10 fold higher, in one embodiment at least 20 fold higher) compared to the in vivo clearance rate for human CCL2 (ml/day/kg) after administration of a bispecific antibody comprising a Fc gamma receptor silenced constant heavy chain domain of human IgGl isotype (or the Fc domain thereof) comprising the mutations L234A, L235A, P329G (Kabat EU numbering), when a pre-formed immune complex consisting of 20mg/kg of each bispecific antibody and O.lmg/kg human CCL2 was administered at a single dose of 10 ml/kg into FcRn transgenic mice.
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L, T307P, K326T and/or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and/or iii) M428L, N434A and/or Y436T (suitable for increasing affinity to FcRn for longer plasma half-life); and/or iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).
  • Kabat EU numbering i) Q311R and/or
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and/or iii) N434A (suitable for increasing affinity to FcRn for longer plasma half- life); and/or iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).
  • Kabat EU numbering i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A (suitable for increasing affinity to FcRn for longer plasma half- life); and iv) Q438R andS440E (suitable for suppressing rheumatoid factor binding).
  • Rabat EU numbering i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L235W, G236N, H268D, Q295L, K326T and A330
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) N434A (suitable for increasing affinity to FcRn for longer plasma half-life); and iii) Q438R and S440E (suitable for suppressing rheumatoid factor binding).
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering)
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L234Y, P238D, T250V, V264I, T307P and/or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and/or iii) M428L, N434A and/or Y436T (suitable for increasing affinity to FcRn for longer plasma half-life); and/or iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life); and iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).
  • Kabat EU numbering i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R and P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A and (suitable for increasing affinity to FcRn for longer plasma half- life); and iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding)
  • such bispecific antibody comprises comprising (independently or in addition to the above described mutations) the following mutations (Kabat EU numbering) i) S354C and T366W in one of the heavy chain constant CH3 domains ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain constant CH3 domains.
  • heterodimerization promoting mutations as described above in the section of Fc domain modifications promoting heterodimerization can used instead of the exemplary knob into holes modifications above.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 175, and a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 176.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of sequence of SEQ ID NO: 175, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 176.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 177, and a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 178.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of sequence of SEQ ID NO: 177, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 178.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 179, and a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 180.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of sequence of SEQ ID NO: 179, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 180.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 181, and a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 182.
  • a specific embodiment of the invention is an (isolated) bispecific antibody comprising a) a first antigen-binding site that (specifically) binds to a first epitope on human CCL2 and b) a second (different) antigen-binding site that (specifically) binds a second (different) epitope on human CCL2 wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of sequence of SEQ ID NO: 181, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 182.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
  • isolated nucleic acid encoding an anti-CCL2 antibody (either bispecific or monospecific) as described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising one or all VL and/or an amino acid sequence comprising one or all VH of the mono- or bispecific antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, a HEK293 cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of making an anti- CCL2 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • an anti-CCL2 cell such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et ak, Nat. Biotech. 24 (2006) 210-215.
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS- 7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F.L. et ak, J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J.P. et ak, Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub, G. et al., Proc. Natl.
  • the invention is based, in part, on the finding that the modified monospecific antibodies as described herein show improved pH dependent binding properties and re therefore especially useful for the generation of the bispecific antibodies of the invention
  • any of the bispecific anti-CCL2 antibodies provided herein is useful for detecting the presence of CCL2 in a biological sample.
  • the term “detecting” as used herein encompasses quantitative or qualitative detection.
  • a biological sample comprises a cell or tissue, such as immune cell or T cell infiltrates and or tumor cells.
  • a bispecific anti-CCL2 antibody for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of CCL2 in a biological sample comprises contacting the biological sample with a bispecific anti-CCL2 antibody as described herein under conditions permissive for binding of the bispecific anti-CCL2 antibody to CCL2, and detecting whether a complex is formed between the bispecific anti-CCL2 antibody and CCL2.
  • Such method may be an in vitro or in vivo method.
  • a bispecific anti-CCL2 antibody is used to select subjects eligible for therapy with a bispecific anti-CCL2 antibody, e.g. where CCL2 is a biomarker for selection of patients.
  • labeled bispecific anti-CCL2 antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No.
  • luciferin 2,3- dihydrophthalazinediones
  • horseradish peroxidase HRP
  • alkaline phosphatase b- galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • compositions of a bispecific anti-CCL2 antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arg
  • exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX ® , Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rhuPH20 HYLENEX ® , Baxter International, Inc.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6, 171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.
  • the formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • bispecific anti-CCL2 antibodies may be used in therapeutic methods.
  • a bispecific anti-CCL2 antibody for use as a medicament is provided.
  • a bispecific anti-CCL2 antibody or use in treating cancer is provided.
  • a bispecific anti-CCL2antibody for use in a method of treatment is provided.
  • the invention provides a bispecific anti-CCL2 antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific anti-CCL2 antibody.
  • the invention provides a bispecific anti-CCL2 antibody inhibits immunesuppresion in tumors and thus makes tumor susceptibel for immuno stimmulatory agenst like anti-PDl, anti-PDL-1 antagonists and the like.
  • one aspect of the is the combination of the bispecific anti-CCL2 antibodies described here with a cancer immunotherapy like anti-PDl, anti-PDL-1 antagonists and the like.
  • cancer as used herein may be, for example, lung cancer, non small cell lung (NSCL) cancer, bronchi oloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis
  • an “individual” according to any of the above embodiments is preferably a human.
  • the invention provides for the use of a bispecific anti-CCL2 antibody in the manufacture or preparation of a medicament.
  • the medicament is for treatment of cancer.
  • the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament.
  • the medicament is for inducing cell mediated lysis of cancer cells
  • the medicament is for use in a method of inducing cell mediated lysis of cancer cells in an individual suffering from cancer comprising administering to the individual an amount effective of the medicament to induce apoptosis in a cancer cell/ or to inhibit cancer cell proliferation.
  • an “individual” according to any of the above embodiments may be a human.
  • the invention provides a method for treating cancer.
  • the method comprises administering to an individual having cancer an effective amount ofbispecific anti-CCL2 antibody.
  • An “individual” according to any of the above embodiments may be a human.
  • the invention provides a method for inducing cell mediated lysis of cancer cells in an individual suffering from cancer.
  • the method comprises administering to the individual an effective amount of a bispecific anti-CCL2 antibody to induce cell mediated lysis of cancer cells in the individual suffering from cancer.
  • an “individual” is a human.
  • a bi specific anti-CCL2 antibody for use in treating inflammatory diseases or autoimmune diseases is provided.
  • the invention provides a bispecific anti-CCL2 antibody for use in a method of treating an individual having an inflammatory disease or autoimmune disease comprising administering to the individual an effective amount of the bispecific anti- CCL2 antibody.
  • the inflammatory diseases or autoimmune disease is an autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder, or a disorder associated with increased numbers or distribution of normal or aberrant tissue resident cells (such as mast cells, macrophages, or lymphocytes) or stromal cells (such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, or endothelia).
  • the disorder is a pulmonary disorder.
  • the pulmonary disorder is associated with granulocytic (eosinophilic and/or neutrophilic) pulmonary inflammation, infection-induced pulmonary conditions (including those associated with viral (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus), bacterial, or fungal (e.g., Aspergillus) triggers.
  • granulocytic eosinophilic and/or neutrophilic
  • infection-induced pulmonary conditions including those associated with viral (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus), bacterial, or fungal (e.g., Aspergillus) triggers.
  • the disorder is an allergen- induced pulmonary condition, a toxic environmental pollutant-induced pulmonary condition (e.g., asbestosis, silicosis, or berylliosis), a gastric aspiration-induced pulmonary condition, or associated with immune dysregulation or an inflammatory condition with genetic predisposition such as cystic fibrosis.
  • a toxic environmental pollutant-induced pulmonary condition e.g., asbestosis, silicosis, or berylliosis
  • a gastric aspiration-induced pulmonary condition e.g., asbestosis, silicosis, or berylliosis
  • a gastric aspiration-induced pulmonary condition e.g., associated with immune dysregulation or an inflammatory condition with genetic predisposition such as cystic fibrosis.
  • the disorder is a physical trauma-induced pulmonary condition (e.g., ventilator injury), emphysema, cigarette-induced emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangiomyomatosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, nosocomial pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), airway exacerbations, and acute respiratory distress syndrome (ARDS)).
  • the inflammatory pulmonary disorder is COPD.
  • the inflammatory pulmonary disorder is asthma.
  • the asthma is persistent chronic severe asthma with acute events of worsening symptoms (exacerbations or flares) that can be life threatening.
  • the asthma is atopic (also known as allergic) asthma, non-allergic asthma (e.g., often triggered by infection with a respiratory virus (e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus) or inhaled irritant (air pollutants, smog, diesel particles, volatile chemicals and gases indoors or outdoors, or even by cold dry air),
  • a respiratory virus e.g., influenza, parainfluenza, rhinovirus, human metapneumovirus, and respiratory syncytial virus
  • inhaled irritant air pollutants, smog, diesel particles, volatile chemicals and gases indoors or outdoors, or even by cold dry air
  • the asthma is intermittent or exercise-induced, asthma due to acute or chronic primary or second-hand exposure to “smoke” (typically cigarettes, cigars, pipes), inhaling or “vaping” (tobacco, marijuana or other such substances), or asthma triggered by recent ingestion of aspirin or related NSAIDS.
  • the asthma is mild, or corticosteroid naive asthma, newly diagnosed and untreated asthma, or not previously requiring chronic use of inhaled topical or systemic steroids to control the symptoms (cough, wheeze, shortness of breath/breathlessness, or chest pain).
  • the asthma is chronic, corticosteroid resistant asthma, corticosteroid refractory asthma, asthma uncontrolled on corticosteroids or other chronic asthma controller medications.
  • the autoimmune disorder, inflammatory disorder, fibrotic disorder, neutrophilic disorder, or eosinophilic disorder is pulmonary fibrosis.
  • the pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF).
  • the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder is esophogitis, allergic rhinitis, non-allergic rhinitis, rhinosinusitis with polyps, nasal polyposis, bronchitis, chronic pneumonia, allergic bronchopulmonary aspergillosis, airway inflammation, allergic rhinitis, bronchiectasis, and/or chronic bronchitis.
  • the autoimmune disorder inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder
  • the arthritis is rheumatoid arthritis.
  • the arthritis is osteoarthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, early arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, psoriatic arthritis, and/or arthritis as a result of injury.
  • the autoimmune disorder, inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder, monocytic disorder, or lymphocytic disorder is a gastrointestinal inflammatory condition.
  • the gastrointestinal inflammatory condition is IBD (inflammatory bowel disease), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused by environmental insults (e.g., caused by or associated with a therapeutic regimen, such as chemotherapy, radiation therapy, etc.), infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, gastritis, gastroenteritis, infectious gastritis or enterocolitis (e.g., Helicobacter pylori-infected chronic active gastritis), and other forms of gastrointestinal inflammation caused by an infectious agent, or indeterminate colitis
  • tissue resident cells such as mast cells, macrophages
  • the autoimmune disorder, inflammatory disorder, or fibrotic disorder is related to sepsis and/or trauma, HIV infection, or idiopathic (of unknown etiology) such as ANCA-associated vaculitides (AAV), granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis), Behcet’s disease, cardiovascular disease, eosinophilic bronchitis, Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), ankylosing spondylitis, dermatomyositis, scleroderma, e.g., systemic scleroderma also called systemic sclerosis, vasculitis (e.g., Giant Cell Arteritis (GCA), also called temporal arteritis, cranial arteritis or Horton disease), myositis, polymyositis, dermatomyositis, polyart
  • the invention provides pharmaceutical formulations comprising any of the bispecific anti-CCL2 antibodies provided herein, e.g., for use in any of the above therapeutic methods.
  • a pharmaceutical formulation comprises any of the bispecific anti-CCL2 antibodies provided herein and a pharmaceutically acceptable carrier.
  • An antibody of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • an antibody of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to 15 mg/kg (e.g. 0.5mg/kg - 10 mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the antibody.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an antibody of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bac
  • a bispecific antibody comprising a first antigen-binding site that
  • the bispecific antibody comprises a) a first polypeptide chain comprising (from N-terminal to C-terminal direction) VH1-CH1-Ll-Hinge-CH2-CH3-L2-VL1-CL wherein,
  • VH1 is a first heavy chain variable domain and VL1 is a first variable light chain domain (both forming together (associating together to form) the first antigen binding site),
  • CHI is a constant heavy chain domain 1
  • L1 is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids)
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • a second polypeptide chain comprising (from N-terminal to C-terminal direction) VH2-CH1-Ll-Hinge-CH2-CH3-L2-VL2-CL wherein, VH2 is a second heavy chain variable domain and VL2 is a second variable light chain domain (both forming together (associating together to form) the second antigen binding site),
  • CHI is a constant heavy chain domain
  • LI is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 5 to 10 amino acids),
  • Hinge is a heavy chain hinge region
  • CH2 is a constant heavy chain domain 2
  • CH3 is a constant heavy chain domain 3
  • L2 is is a polypeptide linker with a length of 5 to 15 amino acids (in one embodiment with a length of 10 to 15 amino acids),
  • CL is a constant light chain domain
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93; or B) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • the VH1 domain comprises the amino acid sequence of SEQ ID NO:73; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID I) i) the VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 72; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO: 94;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • VH1 domain comprises the amino acid sequence of SEQ ID NO: 74; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:91; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • the VH1 domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO: 92; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:93;
  • the Vni domain comprises the amino acid sequence of SEQ ID NO:71; and the VL1 domain comprises the amino acid sequence of SEQ ID NO: 75; and ii) the VH2 domain comprises the amino acid sequence of SEQ ID NO:90; and the VL2 domain comprises the amino acid sequence of SEQ ID NO:94.
  • LI is a polypeptide linker with a length of 9 to 11 amino acids
  • L2 is a polypeptide linker with a length of 9 to 11 amino acids.
  • LI and L2 are polypeptide linkers selected from the group of : GSGGSGGSGG (SEQ ID NO: 183), GSGGGSGGGG (SEQ ID NO: 184), GSGGGGSGGG (SEQ ID NO: 185); GGS GGS GGGG (SEQ ID NO: 186), GGSGGGSGGG (SEQ ID NO: 187), GGSGGGGSGG (SEQ ID NO: 188), GGGSGGSGGG (SEQ ID NO: 189), GGGSGGGSGG (SEQ ID NO: 190), and GGGGSGGSGG (SEQ ID NO: 191.
  • LI is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188), and
  • L2 is a polypeptide linker comprising the amino acid sequence of GGSGGGGSGG (SEQ ID NO: 188).
  • the bispecific antibody according to anyone of the preceding embodiments, wherein the bispecific antibody is not cross-reactive to other CCL homologs, shows 100 time less binding to other CCL homologs (selected from the group of CCL8, CCL7, and CCL13) compared to the binding to CCL2
  • bispecific antibody according to anyone of the preceding embodiments, wherein the bispecific antibody binds to the first and second epitope on human CCL2 in ion-dependent manner.
  • bispecific antibody according to anyone of the preceding embodiments, wherein the bispecific antibody binds to human CCL2 in pH dependent manner and wherein the first antigen binding site and the second antigen binding site both bind to CCL2 with a higher affinity at neutral pH than at acidic pH.
  • bispecific antibody according to anyone of the preceding embodiments, wherein the bispecific antibody binds to human CCL2 with a 10 times higher affinity at pH 7.4, than at pH 5.8.
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Kabat EU numbering) i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and/or iii) N434A (suitable for increasing affinity to FcRn for longer plasma half- life); and/or iv) Q438R and/or S440E (suitable for suppressing rheumatoid factor binding).
  • Kabat EU numbering i) Q311R, and/or P343R (suitable for increasing pi for enhancing uptake of antigen); and/or ii) L235W, G236N
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q
  • Rabat EU numbering i) Q311R and/or P343R (suitable for increasing pi
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and / P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) M428L, N434A and Y436T (suitable for increasing affinity to FcRn for longer plasma half-life); and/ iv) Q438R and S440E(suitable for suppressing rheumatoid factor binding).
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q3
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and/ P343R (suitable for increasing pi for enhancing uptake of antigen); and ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); and iii) N434A (suitable for increasing affinity to FcRn for longer plasma half- life); and / iv) Q438R and S440E (suitable for suppressing rheumatoid factor binding).
  • the constant heavy chain domains CHI, Hinge, CH2 and CH3 are of human IgGl isotype and comprise one or more of the following mutations (Rabat EU numbering) i) Q311R and/ P343R (suitable
  • bispecific antibody according to anyone of the embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 179, and a polypeptide comprising an amino acid sequence that is at least 98%, or 99% identical to the sequence of SEQ ID NO: 180.
  • bispecific antibody according to anyone of the embodiments 1 to 2 and 4 to 7, wherein the bispecific antibody comprises a polypeptide comprising the amino acid sequence of sequence of SEQ ID NO: 181, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 182.
  • a host cell comprising the nucleic acid of embodiment 29.
  • a method of producing an antibody comprising culturing the host cell of embodiment 30 so that the antibody is produced.
  • a pharmaceutical formulation comprising the bispecific antibody according any one of embodiments 1 to 28 and a pharmaceutically acceptable carrier.
  • the bispecific antibody according any one of embodiments 1 to 28 for use as a medicament for use as a medicament.
  • a method of treating an individual having cancer comprising administering to the individual an effective amount of the bi specific antibody according any one of embodiments 1 to 28.
  • a method of treating an individual having an inflammatory or autoimmune disease comprising administering to the individual an effective amount of the bispecific antibody according any one of embodiments 1 to 28.
  • Anti-CCL2 antigen binding moieties (variable regions and hypervariable regions (CDRs) binding to different epitopes:
  • SEQ ID NO: 95 exemplary human kappa light chain constant region
  • SEQ ID NO: 96 exemplary human lambda light chain constant region
  • SEQ ID NO: 97 exemplary human heavy chain constant region derived from IgGl
  • SEQ ID NO: 98 exemplary human heavy chain constant region derived from IgGl with mutations L234A, L235A and P329G (Fcgamma receptor silenced)
  • SEQ ID NO: 99 exemplary human heavy chain constant region derived from IgGl (SGI -IgGl allotype)
  • SEQ ID NO: 100 exemplary human heavy chain constant region derived from IgGl with mutations (SG105-IgGl allotype - Fcgamma receptor silenced)
  • SEQ ID NO: 101 SG1095-exemplary human heavy chain constant region derived from IgGl including the mutations (Kabat EU numbering): -L235W/G236N/H268D/Q295L/A330K/K326T (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR);
  • SEQ ID NO: 102 SG1099-exemplary human heavy chain constant region derived from IgGl including mutations (Rabat EU numbering):
  • CKL02 - SGI 099 (exemplary bispecific CKL02 Crossmab including SGI 099 Fc mutations)
  • CKL02 - SGI 100 (exemplary bispecific CKL02 Crossmab including SGI 100 Fc mutations)
  • CKL03 - SGI 099 (exemplary bispecific CKL03 Crossmab including SGI 099 Fc mutations)
  • CKL03 - SGI 100 (exemplary bispecific CKL03 Crossmab including SGI 100 Fc mutations)
  • SEQ ID NO: 142 exemplary human CCL2 (MCP1) - wild type (wt)
  • SEQ ID NO: 143 exemplary human CCL2 (MCP1) - P8A variant
  • SEQ ID NO: 144 exemplary human CCL2 (MCP1) - T10C variant
  • SEQ ID NO: 145 exemplary human CCL8 (MCP2) - wild type (wt)
  • SEQ ID NO: 146 exemplary human CCL8 (MCP2) - P8A variant
  • SEQ ID NO: 147 exemplary human CCL7 (MCP3) - wild type (wt)
  • SEQ ID NO: 148 exemplary human CCL13 (MCP4)- wild type (wt)
  • SEQ ID NO: 149 exemplary cynomolgus CCL2 - wild type (wt)
  • SEQ ID NO: 150 exemplary mouse CCL2- wild type (wt)
  • SEQ ID NO: 154 GG04 -exemplary human heavy chain constant region derived from IgGl comprising-IgGl allotype sequences) including mutations (Kabat EU numbering): -L234Y/P238D/T250V/V264ET307P/A330K (suitable for increasing affinity to human FcgRIIb and decreasing affinity to other human FcgR); -Q311R/P343R (suitable for increasing isoelectric point (pi) for enhancing uptake of antigen);
  • CKL02 - GG03 (exemplary bispecific CLOK2 Crossmab including GG03 Fc mutations) SEQ ID NO: 163 heavy chain 1- CKL02 - GG03 SEQ ID NO: 164 heavy chain 2- CKL02 - GG03 SEQ ID NO: 165 light chain 1- CKL02 - GG03 SEQ ID NO: 166 light chain 2- CKL02 - GG03
  • CKL02 - GG04 exemplary bispecific CKL02 Crossmab including GG04 Fc mutations
  • P1AF8142 (CKL02 - CB- SGI 095) - (exemplary bispecific CKL02 Contorsbody (CB) comprising only 2 polypetide chains including SGI 095 Fc mutations)
  • P1AF8143 (CKL02 - CB- GG02)- (exemplary bispecific CKL02 Contorsbody (CB) comprising only 2 polypetide chains including GG02 Fc mutations)
  • P1AG5853 (CKL02 - CB- GG02-K447G) - (exemplary bispecific CKL02 Contorsbody (CB) comprising only 2 polypetide chains including GG02 Fc mutations and the mutation K447G)
  • SEQ ID NO: 180 polypeptide chain 2- CKL02 - CB- GG02-KG
  • P1AG8317 (CKL02 - CB- SG1095-K447G) - (exemplary bispecific CKL02 Contorsbody (CB) comprising only 2 polypetide chains including SG1095 Fc mutations and the mutation K447G) SEQ ID NO: 181 polypeptide chain 1- CKL02 - CB- SG1095-KG SEQ ID NO: 182 polypeptide chain 2- CKL02 - CB- SG1095-KG
  • polypeptide linker GGSGGSGG SEQ ID NO: 183 polypeptide linker GSGGSGGSGG SEQ ID NO: 184 polypeptide linker GSGGGSGGGG SEQ ID NO: 185 polypeptide linker GSGGGGSGGG SEQ ID NO: 186 polypeptide linker GGSGGSGGGG SEQ ID NO: 187 polypeptide linker GGSGGGSGGG SEQ ID NO: 188 polypeptide linker GGSGGGGSGG SEQ ID NO: 189 polypeptide linker GGGSGGSGGG SEQ ID NO: 190 polypeptide linker GGGSGGGSGG SEQ ID NO: 191 polypeptide linker GGGGSGGSGG
  • P329G (PGLALA) Bispecific anti-CCL2 Antibodies Alias
  • Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)
  • a transcription unit comprising the following functional elements: the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, a human heavy chain immunoglobulin 5’ -untranslated region (5’UTR), a murine immunoglobulin heavy chain signal sequence, a gene/protein to be expressed (e.g. full length antibody heavy chain or antibody light chain or CCL-2 molecule), and - the bovine growth hormone polyadenylation sequence (BGH pA).
  • P-CMV human cytomegalovirus
  • 5’UTR human heavy chain immunoglobulin 5’ -untranslated region
  • BGH pA bovine growth hormone polyadenylation sequence
  • Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.
  • the expression plasmids for the transient expression of monoclonal antibodies and CCL-2 antigens comprised besides the respective expression cassettes an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.
  • the transcription unit of the respective immunoglobulin HC or LC or CCL-2 molecule comprised the following functional elements: the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, a human heavy chain immunoglobulin 5’ -untranslated region (5’UTR), a murine immunoglobulin heavy chain signal sequence, and the bovine growth hormone polyadenylation sequence (BGH pA).
  • P-CMV human cytomegalovirus
  • intron A a human heavy chain immunoglobulin 5’ -untranslated region
  • 5’UTR human heavy chain immunoglobulin 5’ -untranslated region
  • murine immunoglobulin heavy chain signal sequence a murine immunoglobulin heavy chain signal sequence
  • BGH pA bovine growth hormone polyadenylation sequence
  • the respective antibodies 1A4, 1A5, 1G9, 2F6, CNTQ888, murine and humanized 11K2, ABN912, based on their VH and VL were generated as
  • the recombinant production was performed by transient transfection of HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For the production of monoclonal antibodies, cells were co transfected with plasmids containing the respective immunoglobulin heavy- and light chain. For transfection "293-Fectin" Transfection Reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer’s instructions. Cell culture supernatants were harvested three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (e.g. -80°C).
  • Antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were captured on a Mab Select SuRe resin equilibrated with PBS buffer (10 mMNa2HP04, 1 mMKH2P04, 137 mMNaCl and 2.7 mMKCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0.
  • PBS buffer 10 mMNa2HP04, 1 mMKH2P04, 137 mMNaCl and 2.7 mMKCl, pH 7.4
  • the eluted protein fractions were pooled, neutralized with 2M Tris, pH 9.0 and further purified by size exclusion chromatography using a Superdex 200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Size exclusion chromatography fractions were analysed by CE-SDS (Caliper Life Science, USA) and antibody containing fractions were pooled and stored at - 80°C.
  • Wild type CCL2 can exist as monomer but actually can also form dimers at physiological concentrations. This monomer-dimer equilibrium might be different and has to be carefully taken into account for all in vitro experiments described where different concentrations might be used. To avoid any uncertainties, we generated point mutated CCL2 variants: The P8A variant of CCL2 carries a mutation in the dimerization interface resulting in an inability to form a dimer leading to a defined, pure CCL2 monomer. In contrast, the T10C variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013 Mar 20; 135(11):4325-32).
  • the respective soluble CCL2 protein (wild type, P8A or T IOC variants) was purified from cell culture supernatants by cation exchange chromatography using SP- Sepharose HP (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography. Briefly, sterile filtered cell culture supernatants were diluted with 10 mM KH2P04, pH 5.0 to adjust conductivity ⁇ 4 mS/cm.
  • the diluted supernatant was loaded on SP-Sepharose resin equilibrated with 10 mM KH2P04, pH 5.0, washed with equilibration buffer and eluted using a gradient to 10 mM KH2P04, 1 M NaCl, pH 5.0.
  • the eluted protein fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 16/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by SDS- PAGE and analytical high performance size exclusion chromatography. CCL2 containing fractions were pooled and stored at -80°C.
  • a T200 instalment was mounted with a Biacore Series S Sensor Chip CM5.
  • the system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05 % (w/v) P20).
  • the system was set to 37 °C.
  • the sample buffer was the system buffer, additionally supplemented with 1 mg/ml CMD (Carboxymethyldextran, Fluka).
  • murine monoclonal antibodies were captured on the biosensor by immobilizing 12700 RU polyclonal rabbit anti mouse (RAMIgG, GE Healthcare) antibodies on a Biacore Series CM5 sensor like described above.
  • the sensor was regenerated by a 3 min injection of 10 mM glycine buffer pH 1.7.
  • Antibody clone supernatants were diluted 1 :2 in system buffer and were captured for 1 min at 5 m ⁇ /min. After antibody capturing the system was washed by 2.5-fold concentrated system buffer for 30 sec at 80 m ⁇ /min followed by 2 min baseline stabilization. Analyte kinetics were performed at 30 m ⁇ /min.
  • a T200 instrument was mounted with a Biacore Series S Sensor Chip CM5.
  • the system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.05 % (w/v) P20).
  • the pH of the system buffers was set to pH 8.3, pH 7.9, pH 7.4, pH 7.1, pH 6.7, pH 6.3, pH 5.9, pH 5.5.
  • the system was set to 25 °C.
  • the sample buffer was the system buffer, additionally supplemented with 1 mg/ml CMD (Carboxymethyldextran, Fluka). An antibody capture system was established.
  • CMD Carboxymethyldextran, Fluka
  • Human antibodies were captured as ligands on the sensor surface: ⁇ Human normal IgG as positive control (H-N-IgG, Id.: 11717570, Roche),
  • CCL2 has high homology to CCL 7 (MCP-3), CCL8 (MCP-2), CCL 13 (MCP-4), and these CCL chemokines are able to bind to CCR2, the binding of anti- CCL2 antibodies to these homologs was assessed. Results are shown in Figure 1. With the exception of CNT0888 which was described to have selectivity to CCL2 (Mol Immunol. 2012 Jun; 51(2): 227-33), the other antibodies tested bound to either CCL7 or CCL8 (showed cross-reactivity to either CCL7 or CCL8).
  • Biacore assay method The binding of anti-CCL2 antibodies to the CCL homologs e g. CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3), and CCL 13 (MCP-4) were assessed at 25°C using Biacore T200 instrument (GE Healthcare).
  • Mouse anti human IgG (Fc) (GE Healthcare) was immobilized on each flow cells of a CM4 sensor chip using amine coupling kit (GE Healthcare) according to the recommended settings by the manufacturer.
  • Antibodies and analytes were diluted into ACES pH 7.4 buffer (20 mM ACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20, 0.005%
  • THP-1 human acute monocytic leukemia cell line; ATCC TIB-202 cells were cultivated in RPMI 1640, 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 50 mM b-mercaptoethanol (supplier Thermo Fisher Scientific). On the assay day the cell density was adjusted to 8.33 x 10 5 cells/ml in 25.8 ml assay medium (RPMI 1640 w/o FBS). FLIPR® Calcium Calcium 4 Assay Kit, Cat# R8142,
  • a dye loading solution was prepared by mixing two vials of component A with 20 ml component B (HBSS buffer plus 20 mM HEPES, pH 7.4) according to the instructions of the manufacturer Molecular Devices. 516 m ⁇ 1 M Hepes (final assay concentration: 10 mM) is added followed by 516 m ⁇ 250 mM probenecid (final assay concentration: 2.5 mM). For the stock solution dissolve 65.4 mg probenecid (Sigma P8761) in 465 m ⁇ 1 N NaOH and add 465 m ⁇ lx HBSS (Thermo Fisher Scientific). 25.8 ml loading buffer was mixed with 25.8 ml assay medium with cells sufficient for e.g.
  • the antibody and the ligand solution were prepared. Eight concentrations of each antibody from 30 pg/ml to 0.025 pg/ml (no serial dilution, final concentration in wells) have been tested. Each concentration was tested on two plates. All dilutions were prepared in assay medium as 10-fold concentrated solution. As reference antibody human CCL2/JE/MCP-1 Antibody (R&D Systems Cat# MAB279) was used. Ligand CCL2 (R&D Systems Cat#279-MC-10) was prepared by dissolving 50 pg CCL2 lyophilisate in 500 m ⁇ RPMI 1640 (100 pg/ml) and transferring 400 pi into 10 ml assay medium (4 pg/ml stock solution).
  • As stimulation control ionomycin (Sigma Cat# 1-0634) was used (1 mg ionomycin dissolved in 1340 m ⁇ DMSO (Sigma Cat# D-8779), 1 mM). 10 pi of the ImM stock solution was diluted in 1990 m ⁇ assay medium (5 mM, final assay concentration 500 nM). 100 m ⁇ was pipetted in the corresponding control wells of the polypropylene MTP.
  • the antibody dilutions and CCL2 were preincubated in two V-shape polypropylene 96 well plates. 50 m ⁇ of the 4 pg/ml stock solution CCL2 (final 400 ng/ml CCL2) and 50 m ⁇ of the 10-fold concentrated antibody dilution were pipetted into the well. Plates were incubated for 30 - 60 min at room temperature. After incubation, the cell plate and the compound plate were transferred directly to the FlexStation® 3 (Molecular Devices) read position and the calcium assay was performed as described in the system manual (excitation 485 nm, emission 525 nm). The read out was done at several seconds interval. Results:
  • Table 1 40 ng/ml PMA and 4 mM ionomycin were used as positive controls.
  • Antibody Alias EC50 ⁇ g/ml] EC50 [nM]
  • Monocytes were isolated from peripheral blood of healthy donors by magnetic separation using a commercial kit (Stemcell, cat no. #15068). For blocking of FcyRs, monocytes were pre-incubated with normal human IgG ( Privigen , CSL Behring) at a final concentration of 500pg/ml on ice for 50 min in FACS buffer (PBS + 0.2%BSA). Cells were then centrifuged for 10 min (300xg, 4°C), washed one more time with FACS buffer and stored on ice.
  • IgG Privigen , CSL Behring
  • Anti-CCL2 antibody dilutions (50m1 each) were prepared (in parallel approaches at 4°C and 37°C) in 96 U-bottom wells (BD). Monocytes were split, re suspended in medium (RPMI 1640; 10%FCS; 2mM L-Glutamine) and incubated at 4°C and 37°C, respectively, until further usage. Recombinant CCL2 (50pl; at a final concentration of lOOng/ml) was added to the prepared antibody dilutions (at variable concentrations) both at 4°C and 37°C.
  • IOOmI monocyte suspension (2xl0 5 cells/well) was added to the CCL2/anti-CCL2 mixes at a total volume of 200m1 and cells were incubated at 4°C and 37°C for 1 h 30min before centrifugation at 300xg, 4°C.
  • Antibody Alias EC50 ⁇ g/ml] EC50 [nM]
  • CCR2 + THP1 cells towards a CCL2 gradient was tested as follows.
  • Monocytic THP1 cells (ATCC ⁇ TIB-202TM) were cultured in RPM1 1640 medium (PAN, cat.no. # P04-17500) supplemented with FCS and L-Glutamine. Cells were normally passaged two to three times prior to use in the migration assay and then starved overnight in media with reduced FSC content (1.5% instead of 10% FCS). Cells were counted and incubated with 10 pg/ml normal human IgG (Invitrogen, cat.no. # 12000; to block FcgRs) for 15 minutes at room temperature.
  • anti-CCL2 antibodies (and/or controls) were added to the lower chamber of aHTS Transwell 96well plate system (Coming, cat.no. #3386; 3pm pore size) containing serum-free media with 25 ng/ml rhCCL-2 (R&D Systems, cat.no. #279-MC). Then the insert-plates were stuck into the lower-chamber-plate and 75m1 (1.5xl0 5 cells) of the above mentioned cell-suspension (including the IgG-block) were added with or without 5 pg/ml antibody/isotype into each insert. Plates were covered and incubated over night at 37°C in an C02 incubator (5% C02).
  • the insert-plate was removed and Cell-titer-glo substrate (Promega, cat.no. # G758) was added to each well of lower-chamber-plate to measure viability of migrated cells. After incubation for 1 hour on a shaker with 300 rpm (cover plate sealed), 200 m ⁇ of each well were transferred to a Microfluor black 96well-plate (VWR, cat.no. # 735- 0527) and luminescence was measured (luminescence-reader e.g. Bio-Tek, Tecan).
  • pre-formed immune complexes consisting of anti-CCL2 monoparatopic antibody (20mg/kg) and wild type human CCL2 (O.lmg/kg) were administered at a single dose of 10 ml/kg into the caudal vein of human FcRn transgenic mice (B6 Cg-f c ⁇ /v’ 1111 Dc T g(F CGRT )32Dcr/DcrJ, Jackson Laboratory). Blood was collected 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration. Serum was prepared by centrifuging the blood immediately at 14,000 rpm for 10 minutes in 4°C.
  • the serum was stored at or below -80°C until measurement.
  • the monoparatopic antibodies tested are listed in the Table 3 below.
  • Antibodies with SGI Fc have Fc gamma receptor binding similar to wild-type while antibodies with SG105 Fc are Fc gamma receptor binding silent.
  • SGI IgGl wild type with intact Fc gamma receptor binding
  • SG105 IgGl with no Fc gamma receptor binding
  • the antibody profiles were analyzed by non-compartmental analysis using Phoenix 64 (Pharsight/ Certara).
  • the AUCinf was estimated by linear-log trapezoidal rule extrapolated to infinity. Clearance values are defined as Dose/ AUCinf.
  • Table 3 Clearance values of wild type CCL2 after administration of pre-formed immune complex of anti-CCL2 monospecific antibody (20mg/kg) and wild type human CCL2 (O.lmg/kg) (either IgGl wild type (SGI) or IgGl Fc receptor silenced (SG1051 Clearance Fold Change
  • the concentration of total human CCL2 in mouse serum was measured by ECL.
  • 3ug/mL of anti-CCL2 antibody F7 (Biolegend) or clone MAB679 (R&D Systems) was immobilized onto a MULTI-ARRAY 96-well plate (Meso Scale Discovery) overnight before incubating in blocking buffer for 2 hours at 30°C.
  • Anti-CCL2 MAB679 was used as capture antibody for samples containing humanized 11K2, 1 A4 or 1 A5 antibodies.
  • Anti-CCL2 clone 5D3-F7 was used for samples containing ABN912, CNT0888, 1G9, 2F6H antibodies.
  • Human CCL2 calibration curve samples, quality control samples and mouse serum samples were prepared by diluting in dilution buffer and incubating with excess drug for 30 minutes at 37°C. After that, the samples were added onto anti-CCL2-immobilized plate, and allowed to bind for 1 hour at 30°C before washing. Next, SULFO TAG NHS-ester (Meso Scale Discovery) labelled anti-human Fc (clone: JDC-10, SouthernBiotech) was added and the plate was incubated for 1 hour at 30°C before washing. Read Buffer T (x4) (Meso Scale Discovery) was immediately added to the plate and signal was detected by SECTOR Imager 2400 (Meso Scale Discovery). The human CCL2 concentration was calculated based on the response of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). Measurement of anti-CCL2 antibody concentration in serum by enzyme-linked immunosorbent assay (ELISA)
  • the concentration of anti-CCL2 antibody in mouse serum was measured by ELISA.
  • Anti-human IgG kappa-chain (Antibody Solutions) was dispensed onto a Nunc MaxiSorp plate (Thermofisher) and allowed to stand overnight at 4 degrees C to prepare anti-human IgG-immobilized plates. Calibration curve and samples were prepared with 1% pooled mouse serum. Then, the samples were dispensed onto the anti-human IgG-immobilized plates, and allowed to stand for 1 hour at 30 degrees C. Subsequently, goat anti-human IgG (gamma-chain specific) with HRP conjugate (Southern Biotech) was added to react for 1 hour at 30 degrees C.
  • Chromogenic reaction was carried out using TMB substrate (Life Technologies) as a substrate. After stopping the reaction with 1 N sulfuric acid (Wako), the absorbance at 450 nm was measured by a microplate reader. The concentration in mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).
  • mice mouse cross-reactive 11K2 anti-CCL2 monoparatopic antibodies was administered to mice.
  • humanized 11K2-SG105 Fc gamma receptor binding silent antibodies were intravenously administered at a single dose of 20mg/kg at a single dose of 10 ml/kg into the caudal vein of Balb/c mice.
  • Blood was collected pre-administration, 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days after administration.
  • Serum was prepared by centrifuging the blood immediately at 14,000 rpm for 10 minutes in 4°C. The serum was stored at or below -80°C until measurement.
  • FIG 3 shows the time course of serum total mouse CCL2 concentration and antibody -time profile for humanized 11K2-SG1 and 11K2-SG105 in mice.
  • mice CCL2 concentration of mouse CCL2 in mouse serum was measured by adapting the reagents from a commercially available mouse CCL2 ELISA kit (R&D Systems). The manufacturer’s protocol was followed except for preparation of calibration curve samples. Purified recombinant mouse CCL2 was substituted as the standard instead of the manufacturer’s protein. For samples taken after antibody was injected, calibration curve samples and samples were prepared with 2.5% mouse serum injected antibody spiked in at a concentration of 40 microgram/ml, and incubated for 30 minutes at 37 degrees C. Subsequently, the samples were dispensed onto the anti human CCL2-immobilized plates, and incubated at 30 degrees C for 2 hours. Detection by adding mouse MCP-1 conjugate and incubating for 30 degrees C for 2 hours, followed by substrate and stop solution.
  • Mouse MCP-1 Ultra-Sensitive Kit (Meso Scale Discovery) was used according to the manufacturer’s instructions. No antibody was spiked into the sample before addition to the plate.
  • the concentration of anti-CCL2 antibody in mouse serum was measured by ELISA.
  • Anti-human IgG kappa-chain (Antibody Solutions) was dispensed onto a Nunc MaxiSorp plate (Thermofisher) and allowed to stand overnight at 4 degrees C to prepare anti-human IgG-immobilized plates. Calibration curve and samples were prepared with 1% pooled mouse serum. Then, the samples were dispensed onto the anti-human IgG-immobilized plates, and allowed to stand for 1 hour at room temperature. Subsequently, mouse anti-human IgG HRP (clone JDC-10, Southern Biotech) was added to react for 30 minutes at room temperature.
  • bispecific anti-CCL2 antibodies with two different antigen binding moieties/sites were able to efficiently form immune complexes with CCL2 and clear it from the circulation.
  • Sandwich ELISA was performed to identify antibody pairs that do not compete for binding to human CCL2.
  • 384-well MAXISORP (NUNC) plates were coated with 1 pg/mL of the 7 indicated capture antibodies (Arm 1) and blocked with 2% BSA.
  • Biotinylated (NHS-PECE-Biotin, Pierce) WT human CCL2 (20ng/mL) was incubated with excess amount of the same 7 antibodies (Arm 2) at 1 pg/mL or block buffer for 1 hour at 37 degrees Celsius. After incubation, the mixtures were added to the blocked ELISA plate and incubated for 1 hour at room temperature.
  • Detection of plate bound CCL2 was performed using streptavidin HRP followed by TMB One Component substrate (Lifetech). Signal development was stopped by IN HC1 acid (Wako). The O.D. of wells with no competing antibody was set as 100% signal for each capture antibody. The O.D. of blank wells with no CCL2 added was set as 0% signal. Nine antibody pairs that did not show strong competition for CCL2 binding in both directions were selected as candidates for generation of bispecific Crossmab antibodies.
  • Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. cob plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)
  • a transcription unit comprising the following functional elements:
  • a gene/protein to be expressed e.g. full length antibody heavy chain or MHC class I molecule
  • BGH pA bovine growth hormone polyadenylation sequence
  • the recombinant monoclonal antibody genes encode the respective immunoglobulin heavy and light chains.
  • the expression plasmids for the transient expression monoclonal antibody molecules comprised besides the immunoglobulin heavy or light chain expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.
  • the transcription unit of a respective antibody heavy or light chain comprised the following functional elements:
  • BGH pA bovine growth hormone polyadenylation sequence
  • the recombinant production was performed by transient transfection of HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For the production of monoclonal antibodies, cells were co transfected with plasmids containing the respective immunoglobulin heavy and light chain. For transfection "293-Fectin" Transfection Reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer’s instructions. Cell culture supernatants were harvested three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (e.g. -80°C).
  • IgGl PGLALA structure (non- crossed)
  • Biparatopic anti-CCL2 antibodies containing cell culture supernatants were filtered and purified by up to three chromatographic steps. Depending on the purity of the capture step eluate an ion exchange chromatography step was optionally implemented between capture and polishing step. Biparatopic anti-CCL2 antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden), POROS 50 HS (Thermofisher Scientific) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.
  • sterile filtered cell culture supernatants were captured on a Mab Select SuRe resin equilibrated with PBS buffer (10 mM Na2HP04, 1 mM KH2P04, 137 mM NaCl and 2.7 mM KC1, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0. The eluted protein fractions were pooled and neutralized with 2M Tris, pH 9.0.
  • Ion exchange chromatography as optional second purification step was performed with POROS 50 HS (Thermofisher Scientific), equilibration and wash with 20 mM histidine pH 5.6 and load of diluted capture step eluate a gradient chromatography was done with 20 mM histidine, 0.5MNaCl at pH 5.6. ion exchange chromatography fractions were analyzed by CE-SDS LabChip GX II (PerkinElmer) and Crossmab containing fractions were pooled.
  • Size exclusion chromatography on Superdex 200 was used as second or third purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Size exclusion chromatography fractions were analyzed by CE-SDS LabChip GX II (PerkinElmer) and Crossmab containing fractions were pooled and stored at -80°C.
  • the protein concentration of antibody preparations was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

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Abstract

La présente invention concerne des anticorps anti-CCL2 bispécifiques se liant à deux épitopes différents sur le CCL2 humain, leurs compositions pharmaceutiques, leur fabrication et leur utilisation en tant que médicaments pour le traitement de cancers, de maladies inflammatoires, auto-immunes et ophtalmologiques.
EP22734270.6A 2021-06-18 2022-06-15 Anticorps anti-ccl2 bispécifiques Pending EP4355775A1 (fr)

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US20240360208A1 (en) 2024-10-31
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AU2022295067A1 (en) 2023-12-21

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